Methods and compositions for preventing ischemia reperfusion injury in organs

ABSTRACT

The invention, in some embodiments, relates to compounds and methods for the prevention of ischemia reperfusion injury (IRI) in organs, and in particular to IRI in organs aged 35 years and older. Specific uses include prevention of IRI in native organs in vivo, in reimplantations and in transplantations of donor organs aged 35 years and older. Additional embodiments include the prophylaxis of delayed graft function (DGF) and reduction in the frequency, amount and duration of dialysis in recipients of deceased donor kidney transplantations. The methods entail contacting the organ in vivo or ex vivo with a temporary p53 inhibitor. Novel temporary dsNA p53 inhibitors are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/650,297, filed on Jul. 14, 2017, which is a continuationapplication of U.S. application Ser. No. 15/312,425, filed on Nov. 18,2016 (now abandoned), which is the U.S. National Stage of InternationalApplication No. PCT/US2015/032499, filed on May 27, 2015, which claimsthe benefit of U.S. Provisional Application Ser. No. 62/004,239, filedon May 29, 2014, the contents of each of these applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named “262-PCT1_ST25.txt”, whichis 29 Kbytes in size, and which was created on May 27, 2015 in theIBM-PC machine format, having an operating system compatibility withMS-Windows, and is submitted herewith.

FIELD OF THE INVENTION

The invention, in some embodiments, relates to methods for theprevention of ischemia reperfusion injury (IRI) in organs, and inparticular to IRI in organs aged 35 years and older. Specific usesinclude prevention of IRI in native organs in vivo, in reimplantationsand in transplantations of donor organs aged 35 years and older.Additional embodiments include the prophylaxis of delayed graft function(DGF) and reduction in the frequency, amount and duration of dialysis inrecipients of deceased donor kidney transplantations. The methods entailcontacting the organ in vivo or ex vivo with a temporary p53 inhibitor.Novel temporary dsRNA p53 inhibitors are further provided.

BACKGROUND OF THE INVENTION

Ischemic Reperfusion Injury

Ischemic Reperfusion Injury (IRI) is cellular damage caused to a tissueor organ when blood supply returns to the tissue after a period ofischemia. The absence of blood oxygen and nutrients during the ischemicperiod creates a condition in which the restoration of circulationresults in oxidative damage, including cellular dysfunction, apoptosisand necrosis.

IRI can occur in any body tissue as a result of inter alia surgery,wounds, trauma, obstructions, implantations and transplantations.

Delayed Graft Function

Delayed graft function (DGF) is an important risk factor in adverselyaffecting the outcome of organ transplants, including renal transplantsuch as deceased donor renal transplant (DDRT), particularly in arecipient of a kidney from an Expanded Criteria Donor (ECD) or from aStandard Criteria Donor (SCD) with prolonged cold ischemia time.

Analysis of over 138,000 cases in the UNOS Renal Transplant Registrydatabase, revealed that long-term graft survival (>10 years) hasremained unchanged despite improvements in short term acute rejectionrates (Takada M, et al.; Transplantation. 1997 Dec. 15; 64(11):1520-5.).One of the major contributing factors to poorer long-term outcomesincluding poor graft survival identified in this retrospective reviewwas DGF. DGF is defined by UNOS as the need for dialysis within thefirst seven days after transplantation. The etiology of DGF is not wellunderstood but is undoubtedly multifactorial, in which IRI to the graftthat directly results from the transplantation plays a central role. IRIis an antigen-independent process that is a major risk factor fordevelopment of chronic allograft dysfunction as demonstrated in animalmodels (Goes N, et al., Transplantation. 1995 27; 59(4):565-72; KusakaM, et al., Transplantation. 1999 67(9):1255-61; Takada M, et al;Transplantation. 1997 64(11):1520-5). Ischemic conditions caused byreduced local blood flow to the kidneys during cold storage prior totransplantation followed by oxidative stress after the restoration ofblood supply after the transplantation initiates a chain of events thatcan lead to acute tubular injury. Renal tubular cell dysfunction andapoptotic cell death are the hallmarks of this process (Oberbauer R, etal. J Am Soc Nephrol. 1999 10(9):2006-13; Giral-Classe M, et al. KidneyInt. 1998 54(3):972-8).

Due to the growing disparity between the numbers of candidates awaitingtransplantation and available donor organs, the use of kidneys fromExpanded Criteria Donors (ECD) has been increasing (Organ Procurementand Transplantation Network (OPTN), Scientific Registry of TransplantRecipients (SRTR). 2008 OPTN/SRTR Annual Report: Transplant Data1998-2007 [Internet]. Richmond (Va.): Health Resources and ServicesAdministration's Division of Transplantation (HRSA)/U.S. Department ofHealth & Human Services; 2009 Oct. 7 [cited 2010 April]).

U.S. Pat. Nos. 6,593,353; 6,982,277; 7,008,956 and 7,012,087 relate tothe temporary inhibition of p53 for the treatment of cancer and otherdiseases and disorders.

U.S. Patent Application Publication No. 2006/0069056 to the assignee ofthe present application, is directed to short interfering p53 moleculesand methods of use.

U.S. Pat. Nos. 7,910,566 and 8,148,342 to the assignee of the presentapplication, relate to methods for treating acute kidney injury (AKI)and acute renal failure (ARF), respectively, with short interfering p53molecules.

WO 2010/144336 to the assignee of the present application is directed toa method of treating a subject with chronic kidney disease (CKD)resulting from exposure to a recurring renal insult with a p53inhibitor.

U.S. Patent Application Publication No. US 2010/0222409 and EP Patent NoEP 2170403 to the assignee of the present application relate to a methodof reducing Delayed Graft Function (DGF) in a recipient of a kidneytransplant from a deceased donor using a double-stranded RNA compoundfor down-regulating the expression of a p53 gene.

PCT Patent Application No. PCT/US2013/059349, to the assignee of thepresent application, provides modified double-stranded nucleic acidcompounds for down-regulating the expression of a p53 gene.

There remains an unmet need for a method of prevention or attenuation ofischemic reperfusion injury in native and donor organs.

SUMMARY OF THE INVENTION

The present disclosure is based in part on the surprising and clinicallysignificant finding that recipients of deceased donor kidneys aged 35years or older, wherein the recipients are treated with a temporary p53inhibitor show a greater improvement in clinical outcome compared torecipients of younger deceased donor kidneys and untreated patients. Thepresent inventors have further surprisingly found that temporaryinhibitors of the p53 gene are more effective in providing prophylaxisof DGF in a kidney over the age of about 30 (for example, over the ageof about 35, or over the age of about 40, 45 or 50) and includingkidneys from both Expanded Criteria Donors (ECD) and Standard CriteriaDonors (SCD). Additionally, the treatment of recipients of deceaseddonor kidneys with a temporary p53 inhibitor results in the reduction inthe amount and duration of dialysis post-transplantation compared tountreated recipients.

Aspects and embodiments of the invention are described in thespecification herein below and in the appended claims.

According to a first aspect, there is provided herein a method ofprophylaxis of ischemic reperfusion injury (IRI) of an organ, comprisingcontacting the organ with a temporary p53 gene inhibitor in an amounteffective to provide prophylaxis of IRI in the organ; wherein the organis aged 35 or over. Further provided is a temporary inhibitor of a p53gene for use in prophylaxis of ischemic reperfusion injury (IM), whereinthe inhibitor is for contacting an organ that is 35 years or older atrisk of IRI and use of a temporary inhibitor of a p53 gene for themanufacture of a medicament for providing prophylaxis of ischemicreperfusion injury (IRI), wherein the inhibitor is for contacting anorgan that is 35 years or older at risk of IRI. In some embodiments ofthe method, inhibitor or use wherein the organ is at risk of IRI, theorgan is an organ native to a subject, a reimplanted organ or atransplanted organ.

In some embodiments of the method, inhibitor or use the risk of IRI inthe native organ is imposed by temporary cessation of blood flow to theorgan or by temporary global hypoxia of the organ. The temporarycessation of blood flow is due to, for example, at least one ofthrombosis, vasoconstriction, pressure on blood vessels or removal ofthe organ from the body of a subject with subsequent reimplantation.

The invention, in some embodiments, relates to an organ over the age ofabout 30 or 34; or an organ over the age of about 35; or an organ overthe age of about 36; or an organ over the age of about 37; or an organover the age of about 38; or an organ over the age of about 39; or anorgan over the age of about 40; or an organ over the age of about 41; oran organ over the age of about 42; or an organ over the age of about 43;or an organ over the age of about 44; or an organ over the age of about44; or an organ over the age of about 46; or an organ over the age ofabout 47; or an organ over the age of about 48; or an organ over the ageof about 49; or an organ over the age of about 50; or a donor organ overthe age of about 30 or 34; or a donor organ over the age of about 35; ora donor organ over the age of about 36; or a donor organ over the age ofabout 37; or a donor organ over the age of about 38; or a donor organover the age of about 39; or a donor organ over the age of about 40; ora donor organ over the age of about 41; or a donor organ over the age ofabout 42; or a donor organ over the age of about 43; or a donor organover the age of about 44; or a donor organ over the age of about 44; ora donor organ over the age of about 46; or a donor organ over the age ofabout 47; or a donor organ over the age of about 48; or a donor organover the age of about 49; or a donor organ over the age of about 50; ora deceased Expanded Criteria Donor (ECD). Furthermore, the methodincludes organs that have been preserved entirely by cold storagefollowing removal from the donor and prior to transplantation.

In some embodiments of the method, inhibitor or use the transplant organoriginates from a deceased donor. In various embodiments, the contactingthe organ with the temporary inhibitor comprises administering thetemporary inhibitor to a subject possessing the organ at risk of IRI.The organ at risk may be a native organ of a subject and has never beenremoved from the body of the subject. Alternatively, the organ at riskhas been reimplanted to a subject or transplanted to a subject.

In some embodiments of the method, inhibitor or use the contacting theorgan with the temporary inhibitor comprises contacting the organ withthe temporary inhibitor ex vivo prior to transplantation orreimplantation of the organ to a recipient.

In some embodiments of the method, inhibitor or use the organ at risk ofIRI is 45 years old or older.

In some embodiments of the method, inhibitor, or use prophylaxis of IRIresults in prophylaxis of IRI-associated organ dysfunction or inprophylaxis of IRI-associated delayed graft function.

In some embodiments of the method, inhibitor, or use the organ isselected from the group consisting of a kidney, a liver, a pancreas, aheart, a lung, an intestine, skin, a blood vessel, a brain, a retina,composite tissue, a blood vessel, an ear, a limb; or a part thereof. Insome embodiments the organ is a lung, heart or kidney, preferably akidney.

In various embodiments of the method, inhibitor, or use the organ is akidney graft and wherein prophylaxis of IRI results in prophylaxis ofdelayed graft function (DGF). The prophylaxis of DGF results in thereduction of the amount, intensity and duration of dialytic supportduring at least the first 7 days post-transplant in a dialysis-dependentend stage renal disease (ESRD) patient undergoing deceased donor renaltransplantation. In some embodiments, the prophylaxis of DGF results inat least one of a longer time interval between transplantation and thefirst dialysis treatment post-transplant, a shorter mean duration ofinitial post-transplantation course of dialysis and a higher measuredglomerular filtration rate (mGFR) at the end of the firstpost-transplant month.

In some embodiments of the method, inhibitor or use, the prophylaxis ofIRI results in the reduction of the amount, intensity and/or duration ofdialytic support during the first 30 days, first 60 days, first 120 daysand up to the first 180 days post-transplant in a dialysis-dependent endstage renal disease (ESRD) patient undergoing deceased donor renaltransplantation.

In various embodiments of the method, inhibitor or use the organ, forexample a kidney, is preserved entirely by cold storage followingremoval from the donor and prior to implantation in the recipient.

In various embodiments of the method, inhibitor or use the organ, forexample a kidney is preserved by machine-perfusion for at least aportion of time following removal from the donor and prior toimplantation in the recipient.

In some embodiments of the method, inhibitor or use of prophylaxis ofIRI in a donor kidney, the method further comprises the steps of (a)selecting a recipient having a kidney from a deceased Expanded CriteriaDonor, and (b) administering to the recipient a temporary inhibitor of ap53 gene in an amount effective to provide prophylaxis of DGF in therecipient. In some embodiments, the kidney is from a donor that is not adeceased Expanded Criteria Donor. In alternative embodiments, the kidneyis from a donor that is between the ages of 50 and 59 (inclusive) whodoes not have at least two of the following: a history of high bloodpressure, terminal serum creatinine level greater than 1.5 mg/dl, orcardiovascular cause of brain death. In some embodiments, the kidney isfrom a donor that is not over the age of 60.

In further embodiment of the method, inhibitor or use the prophylaxis ofIRI provides prophylaxis of acute kidney injury (AKI), whereby the AKIresults from at least one of cardiovascular surgery, cardiopulmonarysurgery, renal surgery, acute ureteral obstruction, shock, globalhypoxia and/or exposure to a nephrotoxin.

In a second aspect, provided herein is a method of prophylaxis ofischemic reperfusion injury (IRI) in a donor kidney from a deceaseddonor, comprising contacting the kidney with a temporary inhibitor ofp53 in an amount effective to provide prophylaxis of IRI in the kidney.Further provided is a temporary inhibitor of p53 for use in prophylaxisof ischemic reperfusion injury (IRI) in a donor kidney from a deceaseddonor, wherein the inhibitor is for contacting the kidney. Furtherprovided is use of a temporary inhibitor of a p53 gene for themanufacture of a medicament for providing prophylaxis of ischemicreperfusion injury (IRI) in donor kidney from a deceased donor, whereinthe inhibitor is for contacting the kidney.

In some embodiments of the method, inhibitor or use the prophylaxis ofIRI results in the reduction of the amount, intensity and duration ofdialytic support during the first 180 days post-transplant in adialysis-dependent end stage renal disease (ESRD) patient undergoingdeceased donor renal transplantation.

In some embodiments of the methods, inhibitors or uses disclosedhereinabove, the temporary inhibitor of a p53 gene is selected from thegroup consisting of a small organic molecule, a protein, an antibody orfragment thereof, a peptide, a polypeptide, a peptidomimetic and anucleic acid molecule; or a pharmaceutically acceptable salt or prodrugthereof. The temporary inhibitor of a p53 gene may be a nucleic acidmolecule selected from the group consisting of a single strandedantisense nucleic acid (ssNA), a double-stranded NA (dsNA), a smallinterfering NA (siNA), a short hairpin NA (shNA), a micro RNA (miRNA),an aptamer, and a ribozyme, or a pharmaceutically acceptable salt orprodrug thereof. The nucleic acid molecule may be modified or chemicallymodified. In some embodiments of the method, inhibitor or use, thenucleic acid molecule is a ssNA or a dsNA, comprising one or more of amodified nucleotide, an unmodified nucleotide, a nucleotide analogue andan unconventional moiety.

In some preferred embodiments of the method, inhibitor or use, the dsNAselected from the group consisting of an unmodified dsNA or a chemicallymodified dsNA; or a salt or prodrug thereof. In some embodiments, thedsNA comprises an antisense strand having a nucleic acid sequence setforth in Table 2 (SEQ ID NOS:21-33, 35, 37). In various embodiments, thedsNA comprises an antisense strand sequence 5′ UGAAGGGUGAAAUAUUCUC 3′and a sense strand sequence 5′ GAGAAUAUUUCACCCUUCA 3′.

In some specific embodiments of the method, inhibitor or use, the dsNAmolecule is a synthetic small interfering ribonucleic acid (siRNA)having the structure:

(antisense strand) (SEQ ID NO: 37) 5′ UGAAGGGUGAAAUAUUCUC 3′(sense strand) (SEQ ID NO: 36) 3′ ACUUCCCACUUUAUAAGAG 5′wherein each of A, C, U and G is a ribonucleotide and each consecutiveribonucleotide is joined to the next ribonucleotide by a covalent bond;andwherein alternating ribonucleotides in both the antisense strand and thesense strand are 2′-O-methyl sugar modified ribonucleotides and a2′-O-methyl sugar modified ribonucleotide is present at both the 5′terminus and the 3′ terminus of the antisense strand and an unmodifiedribonucleotide is present at both the 5′ terminus and the 3′ terminus ofthe sense strand. The dsNA may be terminally phosphorylated ornon-phosphorylated at one or more of the 5′ termini and or 3′ termini.In some embodiments, the dsNA is non-phosphorylated at the 5′ terminiand at the 3′ termini. The dsNA molecule is preferably in the form of apharmaceutically acceptable salt, for example a sodium salt.

In various embodiments of the method, inhibitor or use disclosedhereinabove, the prophylaxis of IR injury provides prophylaxis ofwherein the temporary inhibitor of a p53 gene is administered at a doseof about 1.0 mg/kg to about 50 mg/kg, preferable about 10 mg/kg.

In some embodiments of the method, inhibitor or use, the temporaryinhibitor of a p53 gene is formulated as a composition. In someembodiments the temporary inhibitor may be administered as a liquidcomposition comprising a pharmaceutically acceptable carrier.

In some embodiments of the method, inhibitor or use, the compositionfurther comprises a cell targeting moiety. The cell targeting moiety maybe covalently or non-covalently attached to the temporary inhibitor of ap53 gene.

In some embodiments of the method, inhibitor or use, the temporaryinhibitor is administered to the recipient as an injectable compositioncomprising a pharmacologically acceptable aqueous excipient. Thetemporary inhibitor may be administered by intravenous (IV) injection.The intravenous (IV) injection is administered in a single treatment,which may be a single dose or multiple dose. In some embodiments of themethod, inhibitor or use, the single treatment is a single dose ormultiple doses, preferably a single dose. The single treatment is forexample, a single intravenous push (IVP).

In some embodiments of the method, inhibitor or use, the intravenous(IV) injection is administered intraoperatively followingautograft/reimplantation or allograft/transplantation reperfusion. Theintravenous (IV) injection is administered directly into a proximal portof a central venous line or through a peripheral line.

Administration mode and dose of the temporary inhibitor is determined bythe attending physician or hospital staff and will be decided accordingto various factors including the organ, the indication, the overallhealth and age of the subject. In some embodiments, the temporaryinhibitor is administered systemically, subcutaneously, topically, byinhalation, by instillation (lungs). Depending on the target organ, thetemporary inhibitor may be conjugated or formulated, for example, inliposomes, lipoplex, microparticles or nanoparticles.

In some embodiments of the method, inhibitor or use, wherein therecipient received an organ form a donor, the recipient is furtheradministered a medication selected from the group consisting of anantiviral agent, an antifungal agent, an antimicrobial agent, animmunosuppressant agent, and any combination thereof. In someembodiments, the medication is an immunosuppressant agent that is acalcineurin inhibitor. In various embodiments, the immunosuppressantagent is selected from the group consisting of tacrolimus (TAC),mycophenolate mofetil (MMF), mycophenolic acid (MPA), a corticosteroid,a cyclosporine, an azathioprine, a sirolimus, and any combinationthereof. In some embodiments, the immunosuppressant agent is tacrolimus(TAC). The recipient may further be administered an antibody inductiontherapy agent, for example peri-operatively and prior to transplantreperfusion. In various embodiments, the antibody induction therapyagent comprises a polyclonal anti-thymocyte globulin (ATG) or ananti-CD25 (anti-IL-2R) monoclonal antibody.

In some embodiments of the method, inhibitor or use, the inhibitor ispresent in a kit comprising the inhibitor and instructions for use. Forexample, the inhibitor is present within a container in liquid or solidform. The kit may further include a diluent and/or a means foradministration, for example a syringe.

According to an aspect of some embodiments of the invention, there isprovided an inhibitor of a p53 gene for use in prophylaxis of DelayedGraft Function in a recipient of a kidney transplant, wherein therecipient has received the kidney from a donor having a Kidney DonorRisk Index (KDRI) of at least 1.25. According to an aspect of someembodiments of the invention, there is provided the use of an inhibitorof a p53 gene for the manufacture of a medicament for providingprophylaxis of DGF in a recipient of a kidney transplant, wherein therecipient has received the kidney from a donor having a Kidney DonorRisk Index (KDRI) of at least 1.25.

In some embodiments of the method, inhibitor or use described herein,the kidney is from a donor having a KDRI of at least 1.50, at least1.75, at least 2.0, or even at least 2.5. In some embodiments, thekidney is from a donor having a KDRI in the range for 1.25 to 1.50,1.5-175, 1.75-2.0, 2.0-2.5 or even 2.5-3.0.

In some embodiments, the kidney is from a donor having a Kidney DonorProfile Index (KDPI) of at least 70%. In some embodiments, the KDPI isgreater than 75%, greater than 80%, or even greater than 85%.

In some embodiments, the deceased donor kidney is preserved entirely bycold storage following removal from the donor and prior to implantationin the recipient.

In some embodiments, the deceased donor kidney is preserved by machineperfusion for at least a portion of time following removal from thedonor and prior to implantation in the recipient.

In some embodiments, the outcomes of prophylaxis of Delayed GraftFunction comprise at least one of prolonged time-to-firstpost-transplantation dialysis, shorter mean number and duration ofpost-transplantation dialysis and improved measured glomerularfiltration rate (mGFR) during the first post-transplantation month.

In some embodiments, the deceased ECD donor is of age at least about 60years.

In some embodiments, the deceased ECD donor is of age at least about 50years, the donor having at least two conditions selected from the groupconsisting of history of hypertension, terminal serum creatinine levelabove about 1.5 mg/dL and cardiovascular accident as cause of death.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In case of conflict, thespecification, including definitions, takes precedence.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. These terms encompass the terms “consisting of” and“consisting essentially of”.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, the term “Expanded criteria donor (ECD)” refers to akidney donor over the age of 60, or a donor between the ages of 50 and59 (inclusive) with at least two of the following: a history of highblood pressure, terminal serum creatinine level greater than 1.5 mg/dl,or cerebrovascular cause of brain death.

As used herein, the “standard criteria donor (SCD)” is a donor who isunder 50 years of age and suffered brain death from any number ofcauses. This would include donors under the age of 50 who suffer fromtraumatic injuries or other medical problems such as a stroke. Pediatricdonors are considered standard criteria donors; or a donor between theages of 50 and 59 (inclusive) without two or more of the following: ahistory of high blood pressure, terminal serum creatinine level greaterthan 1.5 mg/dl, or cerebrovascular cause of brain death.

As used herein, the term “delayed graft function (DGF)” refers to theneed for dialytic support within 7 days following kidney transfer,excluding allografts that fail within 24 hours post transplantation.

As used herein, the term “Kidney Donor Risk Profile (KDRI)” refers tothe relative risk of post-transplant kidney graft failure (in anaverage, adult recipient) from a particular deceased donor compared tothe median (50th percentile) donor, as defined by the U.S OrganProcurement and Transplantation Network athttp://optn.transplant.hrsa.gov/ContentDocuments/Kidney_Proposal_FAQ.pdf

As used herein, the term “Kidney Donor Profile Index (KDPI)” refers tothe risk of graft failure after kidney transplant, as defined by the U.SOrgan Procurement and Transplantation Network athttp://optn.transplant.hrsa.gov/ContentDocuments/Kidney_Proposal_FAQ.pdf

As used herein, the term “prophylaxis” of DGF refers to prevention orreduction of the intensity and duration of dialytic support, asmanifested, for example, as a longer time interval betweentransplantation and the first dialysis treatment post-transplant,shorter mean duration of the initial post-transplantation course ofdialysis or higher measured glomerular filtration rate at the end of thefirst post-transplant month.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound or molecule (i.e. temporary inhibitor)is an amount sufficient to provide a therapeutic benefit in thetreatment or management of disorders associated with increasedexpression of p53 or to delay or minimize one or more symptomsassociated with disorders associated with increased expression of p53.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound (i.e. inhibitor) is an amount sufficientto prevent, delay the onset or reduce the severity of disordersassociated with increased expression of p53, or one or more symptomsassociated with disorders associated with increased expression of p53 orprevent or delay its recurrence.

As used herein, the term “cold storage” refers to storage at atemperature of about 0° C. or less, for example, storage on ice. Suchstorage reduces the rate of energy consumption, for example, by theorgan.

As used herein, the term “machine-perfusion” refers to storage atbelow-normal body temperatures, together with pump-driven circulation ofa preservation solution through the blood vessels of the kidney. Suchperfusion helps to sustain or replenish residual, intracellular energystores while also reducing the rate at which they are consumed.

An “organ 35 years or older”, refers to a body organ such as a kidney, aliver, a pancreas, a heart, a lung, an intestine, skin, a blood vessel,a brain, a retina, composite tissue, a blood vessel, an ear, a limb; ora part thereof, present in its native host, reimplanted to its nativehost, removed from a donor, or transplanted to a recipient, wherein theage is counted from birth of the host or donor.

A “method of prophylaxis of ischemic reperfusion injury ORD” refers topreventing, attenuating or reducing the damage caused by IRI, forexample, preventing, attenuating or reducing cellular death and/orapoptosis and/or necrosis and/or oxidative stress.

As used herein, “an organ at risk of IRI” refers to an organexperiencing temporary cessation of blood flow or temporary globalhypoxia. In a non-limiting example, a temporary cessation of blood flowmay be due to thrombosis, vasoconstriction, and pressure on bloodvessels for any reason or removal of the organ from the body withsubsequent reimplantation or transplantation.

As used herein, “delayed graft function” or “DGF” refers to organdysfunction following organ transplantation. When referring to a renaltransplant, according to UNOS, DGF is defined as the requirement fordialysis within the first 7 days post-transplant.

As used herein, the term “prophylaxis” of DGF when referring to a renaltransplant, refers to prevention or reduction of the frequency and/orduration of dialytic support, as manifested, for example, as a longertime interval between transplantation and the first dialysis treatmentpost-transplant, shorter mean duration of the initialpost-transplantation course of dialysis or higher measured glomerularfiltration rate at the end of the first post-transplant month.

Temporary Inhibitors of p53

Temporary inhibitors of p53 are intended to reduce the expression orfunction of a p53 gene for a length of time sufficient to evoke atherapeutic or prophylactic effect, for example on an organ or in asubject, without increasing the risk for cancerous growth. A method oftemporary p53 inhibition is disclosed in, inter alia, U.S. Pat. Nos.6,593,353; 6,982,277; 7,008,956 and 7,012,087, incorporated herein byreference in their entirety.

As used herein the term “inhibitor” refers to a compound, which iscapable of reducing (partially or fully) the expression of a gene or theactivity of a product of such gene (mRNA, protein) to an extentsufficient to achieve a desired biological or physiological effect. Forexample, the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10%, 5% or less than that observed in the absence of aninhibitor. Preferably, the inhibitor is a temporary inhibitor thatreversibly reduces p53 expression or activity.

A “temporary” inhibitor of p53 refers to a molecule that exerts itseffect for up to 24 hours, up to 36 hours, up to 48 hours, up to 72hours, up to 96 hours, up to 120 hours or no longer than 120 hours, 7days, 10 days, 20 days or 30 days.

An inhibitor of a “p53 gene” may be a small organic molecule, a protein,an antibody or fragment thereof, a peptide, a polypeptide, apeptidomimetic or a nucleic acid molecule; or a pharmaceuticallyacceptable salt or prodrug thereof.

A small organic molecule may be, for example, pifithrin.

By “nucleic acid aptamer” as used herein is meant a nucleic acidmolecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule, preferably in vivo. The target molecule can beany molecule of interest. For example, the aptamer can be used to bindto a ligand-binding domain of a protein, thereby preventing interactionof the naturally occurring ligand with the protein.

The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody,inter alia. The definition includes polyclonal antibodies or monoclonalantibodies. This term refers to whole antibodies or fragments ofantibodies comprising an antigen-binding domain, e.g. antibodies withoutthe Fc portion, single chain antibodies, miniantibodies, fragmentsconsisting of essentially only the variable, antigen-binding domain ofthe antibody, etc. The term “antibody” may also refer to antibodiesagainst polynucleotide sequences obtained by cDNA vaccination. The termalso encompasses antibody fragments which retain the ability toselectively bind with their antigen, for example a p53 gene product, andare exemplified as follows, inter alia:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule which can be        produced by digestion of whole antibody with the enzyme papain        to yield a light chain and a portion of the heavy chain;    -   (2) (Fab′)2, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′2) is a dimer of two Fab fragments        held together by two disulfide bonds;    -   (3) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (4) Single chain antibody (SCA), defined as a genetically        engineered molecule containing the variable region of the light        chain and the variable region of the heavy chain linked by a        suitable polypeptide linker as a genetically fused single chain        molecule, including a scFv.

CDR grafting may be performed to alter certain properties of theantibody molecule including affinity or specificity. A non-limitingexample of CDR grafting is disclosed in U.S. Pat. No. 5,225,539.

Single-domain antibodies are isolated from the unique heavy-chainantibodies of immunized Camelidae, including camels and llamas. Thesmall antibodies are very robust and bind the antigen with high affinityin a monomeric state. U.S. Pat. No. 6,838,254 describes the productionof antibodies or fragments thereof derived from heavy chainimmunoglobulins of Camelidae.

A monoclonal antibody (mAb) is a substantially homogeneous population ofantibodies to a specific antigen, and is well known in the art.Monoclonal antibodies are obtained by methods known to those skilled inthe art.

A mAb may be of any immunoglobulin class including IgG, IgM, IgE, IgA,and any subclass thereof. A hybridoma producing a mAb may be cultivatedin vitro or in vivo. High titers of mAbs are obtained in vivo forexample wherein cells from the individual hybridomas are injectedintraperitoneally into pristine-primed Balb/c mice to produce ascitesfluid containing high concentrations of the desired mAbs. mAbs ofisotype IgM or IgG may be purified from such ascites fluid, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

By “specific binding affinity” is meant that the antibody binds to a p53polypeptide or fragment thereof with greater affinity than it binds toanother polypeptide under similar conditions.

The term “epitope” is meant to refer to that portion of a moleculecapable of being bound by an antibody which can also be recognized bythat antibody. An “antigen” is a molecule or a portion of a moleculecapable of being bound by an antibody which is additionally capable ofinducing an animal to produce antibody capable of binding to an epitopeof that antigen. An antigen may have one or more than one epitope. Thespecific reaction referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies that may beevoked by other antigens.

Epitopes or antigenic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand have specific three-dimensional structural characteristics as wellas specific charge characteristics.

The antibody may be a human or nonhuman antibody. A nonhuman antibodymay be humanized by recombinant methods to reduce its immunogenicity inman. Methods for humanizing antibodies are known to those skilled in theart.

A mAb or fragment, chimera or humanized antibody thereof may be used asan inhibitor of the p53 gene product, per se, or may be used toconjugate to a temporary inhibitor of a p53 gene. When conjugated to atemporary inhibitor of a p53 gene, the antibody may serve to target anorgan at risk of IRI.

As used herein, the term “peptide” is used broadly to mean peptides,proteins, fragments of proteins and the like. One skilled in the artwill recognize that the peptides disclosed herein may be synthesized aspeptide mimetics. A peptide mimetic or “peptidomimetic”, is a moleculethat mimics the biological activity of a peptide but is not completelypeptidic in nature. The term “peptidomimetic,” as used herein, means apeptide-like molecule that has the activity of the peptide upon which itis structurally based. Such peptidomimetics include chemically modifiedpeptides, peptide-like molecules containing non-naturally occurringamino acids, and peptides and have an activity such as selectivetargeting activity of the peptide upon which the peptidomimetic isderived. A peptidomimetic can include amino acid analogs and can be apeptide-like molecule which contains, for example, an amide bondisostere such as a retro-inverso modification; reduced amide bond;methylenethioether or methylenesulfoxide bond; methylene ether bond;ethylene bond; thioamide bond; trans-olefin or fluoroolefin bond;1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylenebond or another amide isostere. One skilled in the art understands thatthese and other peptidomimetics are encompassed within the meaning ofthe term “peptidomimetic” as used herein. A peptide, protein or fragmentthereof may be used as an inhibitor of the p53 gene product, per se, ormay be used to conjugate to a temporary inhibitor of a p53 gene. Whenconjugated to a temporary inhibitor of a p53 gene, the peptide may serveto target facilitate delivery of the inhibitor to an organ at risk ofIRI.

As used herein a “composition” or “therapeutic composition” refers to apreparation of one or more of the active ingredients with othercomponents such as pharmaceutically acceptable carriers and excipients.The purpose of a therapeutic composition is to facilitate administrationof an active ingredient to a subject.

The term “pharmaceutically acceptable carrier” refers to a carrier or adiluent that does not cause significant irritation to a subject and doesnot substantially abrogate the activity and properties of theadministered active ingredients. An adjuvant is included under thesephrases. The term “excipient” refers to an inert substance added to atherapeutic composition to further facilitate administration of anactive ingredient.

Therapeutic compositions used in implementing the teachings herein maybe formulated using techniques with which one of average skill in theart is familiar in a conventional manner using one or morepharmaceutically acceptable carriers comprising excipients andadjuvants, which facilitate processing of the active ingredients into atherapeutic composition and generally includes mixing an amount of theactive ingredients with the other components. Suitable techniques aredescribed in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition, which is incorporated herein by reference.For example, therapeutic compositions useful in implementing theteachings herein may be manufactured by one or more processes that arewell known in the art, e.g., mixing, blending, homogenizing, dissolving,granulating, emulsifying, encapsulating, entrapping and lyophilizingprocesses.

Therapeutic compositions suitable for implementing the teachings hereininclude compositions comprising active ingredients in an amounteffective to achieve the intended purpose (a therapeutically effectiveamount). Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, for example, isinitially estimated from animal models.

Federal law requires that the use of pharmaceutical compositions in thetherapy of humans be approved by an agency of the Federal government. Inthe United States, enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. section 301-392. Similar approvalis required for most countries. Regulations vary from country tocountry, but individual procedures are well known to those in the artand the compositions and methods provided herein preferably complyaccordingly.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are described herein with reference tothe accompanying figures. The description, together with the figures,makes apparent to a person having ordinary skill in the art how someembodiments of the invention may be practiced.

In the Figures:

FIG. 1 is a graph showing protein levels of ischemia induced activationof p53 in kidneys from young and old rat donors.

FIGS. 2a and 2b are line graphs showing secondary endpoint time to firstpost-transplant dialysis in mITT(EE) population (FIG. 2a ) and in ECD/CSstratum (FIG. 2b ).

FIG. 3 shows a Forest plot demonstrating the impact of QPI-1002treatment on DGF relative risk reduction in graft recipients per donorkidney type.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

Provided herein are compounds and compositions that temporarily inhibitthe p53 gene, and the use of such compounds for prophylaxis of IRI inorgans, a reduction in the amount and duration of dialysis in deceaseddonor kidney transplant recipients and for prevention of Delayed GraftFunction (DGF) in recipients of Expanded Criteria Donor (ECD) kidneys.

Temporary Inhibitors of p53

Temporary inhibitors of p53 are intended to reduce the expression orfunction of a p53 gene for a length of time sufficient to evoke atherapeutic or prophylactic effect, for example in an organ or in asubject, without increasing the risk for cancerous growth. A method oftemporary p53 inhibition is disclosed in, inter alia, U.S. Pat. Nos.6,593,353; 6,982,277; 7,008,956 and 7,012,087, incorporated herein byreference in their entirety.

As used herein the term “inhibitor” refers to a compound, which iscapable of reducing (partially or fully) the expression of a gene or theactivity of a product of such gene (mRNA, protein) to an extentsufficient to achieve a desired biological or physiological effect. Forexample, the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10%, 5% or less than that observed in the absence of aninhibitor. Preferably, the inhibitor is a temporary inhibitor thatreversibly reduces p53 expression or activity.

A “temporary” inhibitor of p53 refers to a molecule that exerts itseffect for up to 24 hours, up to 36 hours, up to 48 hours, up to 72hours, up to 96 hours, up to 120 hours or no longer than 30 days.

An inhibitor of a “p53 gene” may be a small organic molecule, a protein,an antibody or fragment thereof, a peptide, a polypeptide, apeptidomimetic or a nucleic acid molecule; or a pharmaceuticallyacceptable salt or prodrug thereof.

A small organic molecule of may be, for example, pifithrin.

An inhibitor of the p53 gene can be an “aptamer”, which are nucleic acidor peptide molecules that bind to a specific protein target molecule(see, for example, patent documents: Sundaram, et al., Eu. J. Pharm.Sci. 2013, 48:259-271; WO 1992/014843 U.S. Pat. Nos. 5,861,254;5,756,291; 6,376,190.

Aptamers may be used to inhibit target genes or to target otherinhibitors to specific target cells or organs (see for example US2006/0105975).

Aptamers are meant to include “thiophosphate oligonucleotide aptamers,”“thioaptamers” or “TAs”, which are a class of ligand that structurallydiffers from RNA and DNA capable of binding proteins with high (nM)affinity. TAs may also be used to inhibit target genes or as targetingmoieties, per se.

In certain preferred embodiments the compounds that down-regulate orinhibit expression of the p53 gene are nucleic acid molecules (forexample, antisense molecules, short interfering nucleic acid (siNA),short interfering RNA (siRNA), double-stranded NA (dsNA), micro-RNA(miRNA) or short hairpin RNA (shRNA)) that bind a nucleotide sequence(such as an mRNA sequence) or portion thereof, encoding p53, forexample, the mRNA coding sequence (SEQ ID NO:1-7) for human p53,encoding one or more proteins or protein subunits. In variousembodiments the nucleic acid molecule is selected from the groupconsisting of unmodified or chemically modified dsNA compound such as adsRNA, a siRNA or shRNA that down-regulates the expression of a p53gene.

In some embodiments the nucleic acid molecule is a synthetic, unmodifieddouble stranded RNA (dsRNA) compound that down-regulates p53 expression.

In some preferred embodiments the nucleic acid molecule is a synthetic,chemically modified double-stranded RNA (dsRNA) compound thatdown-regulates p53 expression. In certain preferred embodiments, “p53”refers to human p53 gene. In certain preferred embodiments, “targetgene” refers to human p53 gene.

The chemically modified nucleic acid molecules and compositions providedherein exhibit beneficial properties, including at least one ofincreased serum stability, improved cellular uptake, reduced off-targetactivity, reduced immunogenicity, improved endosomal release, improvedspecific delivery to target tissue or cell and increased knockdown/down-regulation activity when compared to corresponding unmodifiednucleic acid molecules.

Nucleic Acid Compounds

In the context of this disclosure, the terms “nucleic acid compound” or“nucleic acid molecule” refer to an oligomer (oligonucleotide) orpolymer (polynucleotide) of deoxyribonucleic acid (DNA) or ribonucleicacid (RNA) or a combination thereof. This term includes compoundscomposed of naturally occurring nucleobases, sugars and covalentinternucleoside linkages.

A “dsRNA” is a small double stranded nucleic acid molecule whichincludes RNA and RNA analogs. A “dsNA” is a small double strandednucleic acid molecule which includes RNA, modified nucleotides and/orunconventional nucleotides. The terms dsRNA and dsNA may be usedinterchangeably.

The term “dsRNA” relates to two strands of anti-parallel polyribonucleicacids held together by base pairing. The two strands can be of identicallength or of different lengths provided there is enough sequencehomology between the two strands that a double stranded structure isformed with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%complementarity over the entire length. According to one embodiment,there are no nucleotide overhangs for the dsRNA molecule. According toanother embodiment, the dsRNA molecule comprises overhangs, which may beselected from nucleotide overhangs, non-nucleotide overhangs or acombination thereof. According to other embodiments, the strands arealigned such that there are between 1-10 bases, preferably between 1-6bases at least at the end of the strands, which do not align such thatan overhang of 1-10 residues occurs at one or both ends of the duplexwhen strands are annealed.

In some embodiments, the dsRNA in the present application is between 15and 100 bp, between 15 and 50 bp, between 15 and 40 bp, between 15 and30, or between 15 and 25 bp. In a further embodiment, the 5′ and/or 3′ends of the sense and/or antisense strands of the dsRNA comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide overhangs. In someembodiments, the dsRNA comprises between 1-6 nucleotide overhangs on the5′ and/or 3′ ends of the sense and/or antisense strands. In anotherembodiments, the ends of the dsRNA are blunt.

The term “siRNA” relates to small inhibitory dsRNA (generally between15-25 bp) that may interact with the RNA interference (RNAi) machineryand induce the RNAi pathway. Typically, siRNA are chemically synthesizedas 15-25 mers, preferably comprising a central 15-19 bp duplex regionwith or without symmetric 2-base or more 3′ overhangs on the termini.

The strands of a double stranded interfering RNA (e.g. siNA and siRNA)may be connected to form a hairpin or stem-loop structure (e.g. ashRNA). Thus, as mentioned the RNA silencing agent of some embodimentsof the invention may also be a short hairpin RNA (shRNA).

The term “shRNA”, as used herein, refers to a RNA molecule having astem-loop structure, comprising a first and second region ofcomplementarity sequence, the degree of complementarity and orientationof the regions being sufficient such that base pairing occurs betweenthe regions, the first and second regions being sufficient such thatbase pairing occurs between the regions, the first and second regionsbeing bound by a loop region, the loop resulting from lack of basepairing between nucleotides (or nucleotide analogs) within the loopregion.

As used herein the term “modified nucleotide” refers to a nucleotidecomprising at least one modification which may be a sugar modification,a nucleobase modification or an internucleotide linkage modification(between said nucleotide and a consecutive nucleotide) or a combinationthereof. Such modified nucleotides are often preferred over thenaturally occurring forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a nucleic acidtarget and enhanced nuclease stability.

Internucleotide Linkage Modifications:

The naturally occurring internucleoside linkage that makes up thebackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Thenucleic acid compounds according to some embodiments of the inventionmay comprise at least one modified (non-naturally occurring)internucleotide linkage. Modified internucleoside linkages may includeinternucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom.

Non-limiting examples of modified internucleoside linkages containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-allylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate(PACE) and thiophosphonoacetate, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

Non-limiting examples of modified internucleotide linkages that do notinclude a phosphorus atom therein include a short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane linkages; sulfide, sulfoxide and sulfonelinkages; formacetyl and thioformacetyl linkages; methylene formacetyland thioformacetyl linkages; riboacetyl linkages; alkene containinglinkages; sulfamate linkages; methyleneimino and methylenehydrazinolinkages; sulfonate and sulfonamide linkages; amide linkages; and otherlinkages having mixed N, O, S and CH₂ component parts.

Non-limiting examples of heteroatom internucleoside linkages include—CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene (methylimino)or MMI linkage), —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂— (wherein the naturally occuring phosphodiesterinternucleotide linkage is represented as —O—P(═O)(OH)—O—CH₂—).

Sugar Modifications:

Sugar moieties in nucleic acid compounds disclosed herein may include2′-hydroxylpentofuranosyl sugar moiety without any modification.Alternatively, nucleic acid compounds of the invention may contain oneor more substituted or otherwise modified sugar moieties. A preferredposition for a sugar substituent group is the 2′-position not usuallyused in the native 3′ to 5′-internucleoside linkage. Other preferredpositions are the 3′ and the 5′-termini. 3′-sugar positions are open tomodification when the linkage between two adjacent sugar units is a2′-5′-linkage. Preferred sugar substituent groups include: —OH; —F;—O-alkyl, —S-alkyl, or —N— alkyl; —O-alkenyl, —S-alkenyl, or —N-alkenyl;—O-alkynyl, —S-alkynyl or —N-alkynyl; or —O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10alkyl or C2 to C10 alkenyl and alkynyl, C1 to C10 lower alkyl,substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl (e.g.-propargyl, -propyl, -ethynyl, -ethenyl and propenyl). Non-limitingexamples of sugar modification include methoxy (—O—CH₃), methylthio(—S—CH₃), —OCN, aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂),—O-allyl (—O—CH₂—CH═CH₂), —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃,—O(CH₂)_(n)NH₂, —O(CH₂)_(n)CH₃, —O(CH₂)_(n)ONH₂, and—O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10,2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃), 2′-dimethylaminooxyethoxy(2′-O(CH₂)₂ON(CH₃)₂), —N-methylacetamide (2′-O—CH₂—C(═O)—N(H)CH₃), —F,—Cl, —Br, —I, —CN, —SOCH₃, —SO₂CH₃, —ONO₂, —NO₂, —N₃, —NH₂, imidazole,carboxylate, thioate, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino and substituted silyl.

The 2′-sugar substituent groups described supra may be incorporated inthe arabino (up) position or ribo (down) position. An example of2′-arabino modification is 2′-F (2′-F-arabino modified nucleotide istypically referred to as fluoroarabimo nucleic acid (FANA)). Accordingto some embodiments, sugar moieties may be modified such as,2′-deoxy-pentofuranosyl sugar moiety.

In some preferred embodiments, the modified nucleotide comprises atleast one 2′-O-methyl sugar modified ribonucleotide.

Similar modifications to the ones described supra may also be made atpositions other than the 2′ position of the sugar moiety, particularlyat the 3′ position of the sugar of a 3′ terminal nucleoside or in a2′-5′ linked nucleotides and the 5′ position of a 5′ terminalnucleotide.

Nucleobase Modifications:

The nucleic acid compounds disclosed herein may comprise “unmodified” or“natural” nucleobases including the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Alternatively, nucleic acid compounds of the invention may contain oneor more substituted or otherwise modified nucleobase. Non-limitingexamples of nucleobase modifications include 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl adenine, 6-methyl guanine, 2-propyl adenine, 2-propyl guanineand other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynyl cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo adenine, 8-halo guanines, 8-aminoadenine, 8-amino guanine, 8-thiol adenine, 8-thiol guanine, 8-thioalkyladenine, 8-thioalkyl guanine, 8-hydroxyl adenine, 8-hydroxyl guanine andother 8-substituted adenines and guanines, 5-halo (e.g. 5-bromo) uracil,5-halo (e.g. 5-bromo) cytosine, 5-trifluoromethyl uracil,5-trifluoromethyl guanine and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine.Modified nucleobases moieties may also include those in which the purineor pyrimidine base is replaced with other heterocycles, for example8-azaguanine, 8-azaadenine, 7-deaza-guanine, 7-deaza-adenine,3-deazaguanine and 3-deazaadenine. Additional examples includenucleobases having non-purinyl and non-pyrimidinyl bases such as2-aminopyridine, 2-pyridone and triazine.

Nucleotide Analogues:

The nucleic acid compounds, according to some embodiments of theinvention, may comprise one or more nucleotide analogues. The term“Nucleotide analogues” alternatively referred to as “nucleotidemimetics” as used herein refers to nucleotides wherein the furanose ringor the furanose ring and the internucleotide linkage are replaced withalternative groups. The nucleobase moiety (modified or unmodified) ismaintained.

Non-limiting examples of nucleotide analogues include a peptide nucleicacid (PNA), in which the sugar-backbone of a nucleotide is replaced withan amide containing backbone, in particular an aminoethylglycinebackbone; a morpholino nucleic acid, in which the furanose ring isreplaced with a morpholine ring; a cyclohexenyl nucleic acid (CeNA), inwhich the furanose ring is replaced with a cyclohenyl ring; a nucleicacid comprising bicyclic sugar moiety (BNAs), such as “Locked NucleicAcids” (LNAs) in which the 2′-hydroxyl group of the ribosyl sugar ringis linked to the 4′ carbon atom of the sugar ring thereby forming a2′-C,4′-C-oxymethylene linkage to form the bicyclic sugar moiety, a2′-O,4′-ethylene-bridged nucleic acid (ENA) and the like; a threosenucleic acid in which the hydroxylpentofuranosyl sugar moiety isreplaced with threose sugar moiety; an arabino nucleic acid (ANA) inwhich the ribose sugar moiety is replaced with arabinose sugar moiety;an unlocked nucleic acid (UNA), in which the ribose ring is replacedwith an acyclic analogue, lacking the C2′-C3′ bond; a mirror nucleotidein which the typical D-ribose ring is replaced with a L-ribose ring,thus forming a nucleotide which is a mirror image of natural nucleotideand a nucleotide comprising a 2′-5′ linkage, in which the typical 3′ to5′ internucleotide linkage is replaced with a 2′ to 5′ linkage(preferably a 2′-5′ phosphate based internucleotide linkage). It is tobe emphasized that the nucleotide analogues described, may be furthermodified as described above for “modified nucleotide”.

The nucleic acid compound according to some embodiments of the inventionmay further comprise at least one unconventional moiety. The term“unconventional moiety” as used herein refers to as an “abasicnucleotide” or an “abasic nucleotide analog”. Such abasic nucleotideencompasses sugar moieties lacking a base or having other chemicalgroups in place of base at the 1′ position. The abasic nucleotide maycomprise an abasic ribose moiety (unmodified or modified as describedsupra) or an abasic deoxyribose moiety (unmodified or modified).Additionally, the abasic nucleotide may be a reverse abasic nucleotide,e.g., where a reverse abasic phosphoramidite is coupled via a 5′ amidite(instead of 3′ amidite) resulting in a 5′-5′ phosphate linkage. The term“abasic nucleotide analog encompasses any nucleotide analog as definedabove, wherein the sugar moiety is lacking a base or having otherchemical groups in place of base at the 1′ position.

Terminal Modifications:

Modifications can be made at terminal phosphate groups. Non-limitingexamples of different stabilization chemistries can be used, for exampleto stabilize the 3′-end of nucleic acid sequences, include[3-3′]-inverted deoxyribose; deoxyribonucleotide;[5′-3′]-3′-deoxyribonucleotide; [5′-3′]-ribonucleotide;[5′-3′]-3′-O-methyl ribonucleotide; 3′-glyceryl;[3′-5′]-3′-deoxyribonucleotide; [3′-3′]-deoxyribonucleotide;[5′-2′]-deoxyribonucleotide; and [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone structures indicated, thesestructures can be combined with different internucleotide linkagemodifications, sugar modifications and/or nucleobase modifications asdescribed above.

The nucleic acid compounds according to some embodiments disclosedherein may comprise blunt ends (i.e., ends do not include anyoverhanging nucleotides). Alternatively, the nucleic compounds of theinvention may comprise at least one overhang, said overhangs may beselected from the group consisting of nucleotide overhangs (e.g.3′-terminal nucleotide overhangs) and non-nucleotide overhangs.

In particular embodiments, chemically modified dsNA compounds thattarget p53, compositions and kits comprising same and methods of usethereof in the treatment of a condition or pathology involving apoptosis(programmed cell death), are provided herein. In particular embodimentsthe invention, relates to use of such compounds for prophylaxis ofDelayed Graft Function (DGF) in a recipient of a kidney from a deceasedExpanded Criteria Donor (ECD), including as a kidney that has beenpreserved entirely by cold storage following removal from the donor andprior to implantation in the recipient.

In some embodiments the nucleic acid compounds that target anddown-regulate the p53 gene are having oligonucleotide sequences (SEQ IDNOS: 8-37). In some embodiments pharmaceutically acceptable salts ofsuch compounds are used. In some embodiments of nucleic acid compoundshaving a double-stranded structure, the oligonucleotide sequence of oneof the strands is selected from one of SEQ ID NOS: 8-20, 34 and 36 andthe oligonucleotide sequence of the other strand is selected from one ofSEQ ID NOS: 21-33, 35 and 37. For example, SEQ ID NO:21; SEQ ID NO:22;SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27;SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32;SEQ ID NO:33; SEQ ID NO:35; or SEQ ID NO:37.

In some embodiments, the inhibitors of p53 are nucleic acid molecules,or pharmaceutically acceptable salts of such molecules, having adouble-stranded structure in which (a) the nucleic acid molecule is aduplex which includes a sense strand and a complementary antisensestrand; (b) each strand of the nucleic acid molecule is 19 nucleotidesin length; (c) a 19 nucleotide sequence of the antisense strand iscomplementary to a consecutive sequence of a mRNA encoding mammalian p53(e.g., SEQ ID NOS: 1-7) or portion thereof; and (d) the sense strand andantisense strand are selected from the oligonucleotide sequences setforth in Table 1 below (SEQ ID NOS: 8-37).

TABLE 1Selected sense strand and antisense strand oligonucleotide sequences for nucleic acid compounds targeting p53 SEQ ID SEQ ID NOSense strand (5′ > 3′) NO Antisense strand (5′ > 3′)  8 5′CAGACCUAUGGAAACUACU 3′ 21 5′ AGUAGUUUCCAUAGGUCUG 3′  9 5′GGAUGUUUGGGAGAUGUAA 3′ 22 5′ UUACAUCUCCCAAACAUCC 3′ 10 5′GACUCAGACUGACAUUCUA 3′ 23 5′ UAGAAUGUCAGUCUGAGUC3′ 11 5′GGGUUGGUAGUUUCUACAA 3′ 24 5′ UUGUAGAAACUACCAACCC 3′ 12 5′GGGAUGUUUGGGAGAUGUA 3′ 25 5′ UACAUCUCCCAAACAUCCC 3′ 13 5′GGAUCCACCAAGACUUGUA 3′ 26 5′ UACAAGUCUUGGUGGAUCC 3′ 14 5′GAGGGAUGUUUGGGAGAUA 3′ 27 5′ UAUCUCCCAAACAUCCCUC 3′ 15 5′GGGCCUGACUCAGACUGAA 3′ 28 5′ UUCAGUCUGAGUCAGGCCC 3′ 16 5′GACUCAGACUGACAUUCUU 3′ 29 5′ AAGAAUGUCAGUCUGAGUC 3′ 17 5′GCAUUUGCACCUACCUCAA 3′ 30 5′ UUGAGGUAGGUGCAAAUGC 3′ 18 5′GGAUGUUUGGGAGAUGUAU 3′ 31 5′ AUACAUCUCCCAAACAUCC 3′ 19 5′GGGCCUGACUCAGACUGAU 3′ 32 5′ AUCAGUCUGAGUCAGGCCC 3′ 20 5′CAGACCUAUGGAAACUACA 3′ 33 5′ UGUAGUUUCCAUAGGUCUG 3′ 34 5′CCGAGUGGAAGGAAAUUUG 3′ 35 5′ CAAAUUUCCUUCCACUCGG 3′ 36 5′GAGAAUAUUUCACCCUUCA 3′ 37 5′ UGAAGGGUGAAAUAUUCUC 3′

All positions given in Table 1 are 5′>3′ on the sense strand and on theantisense strand.

In other embodiments, the inhibitors of p53 are nucleic acid compounds(e.g., dsNA molecules), or pharmaceutically acceptable salts of suchcompounds, in which (a) the nucleic acid molecule is a duplex whichincludes a sense strand and a complementary antisense strand; (b) eachstrand of the nucleic acid molecule is 19 nucleotides in length; (c) a19 nucleotide sequence of the antisense strand is complementary to aconsecutive sequence of a mRNA encoding mammalian p53 (e.g., SEQ ID NOS:1-7) or portion thereof; and (d) the sense strand and antisense strandcomprise sequence pairs set forth in Table 2 below.

TABLE 2Selected pairs of sense and antisense strands for generating double-stranded nucleic acid compounds targeting p53 SEQ ID SEQ ID Pair Name NOSense strand (5′ > 3′) NO Antisense strand (5′ > 3′) p53_1 36 5′GAGAAUAUUUCACCCUUCA 3′ 37 5′ UGAAGGGUGAAAUAUUCUC 3′ p53_13  8 5′CAGACCUAUGGAAACUACU 3′ 21 5′ AGUAGUUUCCAUAGGUCUG 3′ p53_34  9 5′GGAUGUUUGGGAGAUGUAA 3′ 22 5′ UUACAUCUCCCAAACAUCC 3′  9 5′GGAUGUUUGGGAGAUGUAA 3′ 31 5′ AUACAUCUCCCAAACAUCC 3′ p53_35 10 5′GACUCAGACUGACAUUCUA 3′ 23 5′ UAGAAUGUCAGUCUGAGUC 3′ p53_36 11 5′GGGUUGGUAGUUUCUACAA 3′ 24 5′ UUGUAGAAACUACCAACCC 3′ p53_37 12 5′GGGAUGUUUGGGAGAUGUA 3′ 25 5′ UACAUCUCCCAAACAUCCC 3′ p53_38 13 5′GGAUCCACCAAGACUUGUA 3′ 26 5′ UACAAGUCUUGGUGGAUCC 3′ p53_39 14 5′GAGGGAUGUUUGGGAGAUA 3′ 27 5′ UAUCUCCCAAACAUCCCUC 3′ p53_40 15 5′GGGCCUGACUCAGACUGAA 3′ 28 5′ UUCAGUCUGAGUCAGGCCC 3′ p53_41 16 5′GACUCAGACUGACAUUCUU 3′ 29 5′ AAGAAUGUCAGUCUGAGUC 3′ p53_42 17 5′GCAUUUGCACCUACCUCAA 3′ 30 5′ UUGAGGUAGGUGCAAAUGC 3′ p53_43 18 5′GGAUGUUUGGGAGAUGUAU 3′ 31 5′ AUACAUCUCCCAAACAUCC 3′ 18 5′GGAUGUUUGGGAGAUGUAU 3′ 22 5′ UUACAUCUCCCAAACAUCC 3′ p53_44 19 5′GGGCCUGACUCAGACUGAU 3′ 32 5′ AUCAGUCUGAGUCAGGCCC 3′ 19 5′GGGCCUGACUCAGACUGAU 3′ 28 5′ UUCAGUCUGAGUCAGGCCC 3′ p53_45 20 5′CAGACCUAUGGAAACUACA 3′ 33 5′ UGUAGUUUCCAUAGGUCUG 3′ 20 5′CAGACCUAUGGAAACUACA 3′ 21 5′ AGUAGUUUCCAUAGGUCUG 3′

All positions given in Table 2 are 5′>3′ on the sense strand and on theantisense strand.

In preferred embodiments the sense strand and the antisense strand ofthe double-stranded nucleic acid molecule are selected from the groupconsisting of a sense strand SEQ ID NO: 36 and an antisense strand SEQID NO: 37; a sense strand SEQ ID NO: 16 and an antisense strand SEQ IDNO: 29; a sense strand SEQ ID NO: 19 and an antisense strand SEQ ID NO:32; and a sense strand SEQ ID NO: 19 and an antisense strand SEQ ID NO:28.

QP-1002

QPI-1002 (also known as “I5NP”, CAS Number 1231737-88-4) havingmolecular weight 12,319.75 Daltons (protonated form), QPI-1002 SodiumSalt: 13,111.10 Daltons (sodium salt) is nuclease-resistant, chemicallymodified, synthetic, double-stranded (19-base pair) RNA oligonucleotidedesigned to temporarily inhibit the expression of the pro-apoptoticgene, p53, via activation of the RNA interference (RNAi) pathway. Thesodium salt of QPI-1002 has the molecular formula:C₃₈₀H₄₄₈O₂₆₂N₁₄₀P₃₆Na₃₆. The RNA duplex is partially protected fromnuclease degradation using a modification on the 2′ position of theribose sugar.

The Structure of QPI-1002 is as Follows:

(antisense strand) (SEQ ID NO: 37) 5′ UGAAGGGUGAAAUAUUCUC 3′(sense strand) (SEQ ID NO: 36) 3′ ACUUCCCACUUUAUAAGAG 5′wherein each of A, C, U and G is a ribonucleotide and each consecutiveribonucleotide is joined to the next ribonucleotide by a covalent bond;and wherein alternating ribonucleotides in both the antisense strand andthe sense strand are 2′-O-methyl sugar modified ribonucleotides and a2′-O-methyl sugar modified ribonucleotide is present at both the 5′terminus and the 3′ terminus of the antisense strand and an unmodifiedribonucleotide is present at both the 5′ terminus and the 3′ terminus ofthe sense strand. Such that in the antisense strand each of the first,third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth,seventeenth and nineteenth ribonucleotide is a 2′-O-Methyl sugarmodified ribonucleotide; and in the sense strand each of the second,fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth andeighteenth ribonucleotide is a 2′-O-Methyl sugar modifiedribonucleotide.

p53 protein is activated as a consequence of the acute renal tubular(ischemia-reperfusion) injury that can occur in donor kidneystransplanted following hypothermic preservation, particularly prolongedhypothermic preservation, such as for periods of 26 hours or more, andafter removal of patients from cardiopulmonary bypass following majorcardiac surgery, leading to the induction of apoptosis/programmed celldeath. The temporary inhibition of p53 expression by QPI-1002 affordsproximal tubular epithelial cells time to repair cellular damage and,therefore, avoid induction of apoptosis. Temporarily blocking inductionof apoptosis has been shown by the present inventors reduce theseverity, frequency or duration of reperfusion injury followingprolonged ischemia.

The administered dose of the temporary inhibitor of p53 must beeffective to achieve prophylaxis, including but not limited to improvedsurvival rate or more rapid recovery, or improvement or attenuation orprevention of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The compoundsdisclosed herein can be administered by any of the conventional routesof administration. It should be noted that the compound can beadministered as the compound or as pharmaceutically acceptable salt andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. Liquid forms may be prepared for injection, the termincluding subcutaneous, transdermal, intravenous, intramuscular,intrathecal, and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organiccosolvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In addition, under certaincircumstances the compositions for use in the novel treatments of thepresent invention may be formed as aerosols, for intranasal and likeadministration. The patient being treated is a warm-blooded animal and,in particular, mammals including man. The pharmaceutically acceptablecarriers, solvents, diluents, excipients, adjuvants and vehicles as wellas implant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients of the invention and they include liposomes, lipidatedglycosaminoglycans and microspheres. Many such implants, deliverysystems, and modules are well known to those skilled in the art.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Example 1: Ischemia-Reperfusion Induced p53 Activation in Kidneys fromRat Donors

Kidneys were harvested from young (3-month old) and old (14-month old)SD rats (n=4-6) and subjected to cold ischemia (CI) for 5 hours. Onekidney from each animal was processed for protein extraction whereas thesecond kidney was transplanted into 3-month old syngeneic rats. Thetransplanted kidneys were harvested 24 hours after the surgery and theresultant onset of ischemia-reperfusion injury (IR) and also processedfor protein extraction. Protein extracts were analyzed in ELISA for p53expression levels.

Results: In FIG. 1, the Y-axis shows arbitrary units corresponding top53 protein levels measured in ELISA. FIG. 1 shows that the p53 proteinlevel is significantly increased in transplanted kidneys from olderrats, compared to transplanted kidneys from young rats (3 months old).Note that following p53 activation occurring via a variety ofpost-translational modifications, its steady-state levels are increaseddue to marked protein stabilization.

Example 2: Use of an Inhibitor of a p53 Gene for Prophylaxis of IschemicReperfusion Injury (IRI) and Delayed Graft Function (DGF) in a KidneyTransplantation Recipient

A placebo-controlled, randomized, prospective, double-blind,multicenter, phase II, dose-escalation study of the clinical activity ofQPI-1002 (10 mg/kg single bolus IV dose given at 30 minutes after thereperfusion) for prophylaxis of delayed graft function in ESRDdialysis-dependent patients undergoing deceased donor kidneytransplantation as well as the safety and PK of QPI-1002 in thesepatients.

The primary objectives of the study were 1) to assess the efficacy ofQPI-1002 in the prevention of DGF and 2) to further assess the safety ofa single-dose IV bolus infusion of QPI-1002 in high-risk patientsfollowing deceased donor renal transplantation.

The primary endpoint was the incidence of DGF, whereas DGF was definedin the protocol as the necessity for dialysis during the first 7 daysfollowing the transplantation. The secondary endpoints includedparameters measuring dialysis severity in dialyzed patients, kidneyfunction in non-dialyzed patients immediately post-transplant (within 5days) as well as kidney function at an intermediate-term observationpoint, i.e. at 30 days after transplantation.

Materials and Methods

Test product: QPI-1002 was provided by Quark Pharmaceuticals Inc.,Fremont Calif., formulated as a preservative free, sterile solutionformulated in phosphate buffered saline. The product was filled intoclear Type I glass vials sealed with Teflon-coated butyl rubber stopperswith aluminum flip-off overseals. Each vial was provided for single use.Vials were stored at 2−8° C., protected from light. The solution waswarmed to room temperature prior to use.

QPI-1002 was administered via bolus intravenous injection at a dose of10 mg/kg at about 30 minutes following completion of surgery and removalof the patient from the cardiopulmonary bypass machine, or followingreperfusion of the transplanted kidney.

Cold storage: Following removal from the donor, kidneys preserved bycold storage were flushed with cold preservative solution and placed ina sterile bag immersed in the solution. The sterile bag was placedinside an additional bag containing crushed ice.

Machine-perfusion: Following removal from the donor, kidneys preservedby machine-perfusion were connected to a perfusion device configured tocontinuously pump perfusion fluid through the organ. non-limitingexamples of commonly used devices for machine perfusion of donor kidneysinclude the Waters RM3 (IGL/Waters Medical Systems, Rochester Minn.) andthe LifePort kidney transporter (Organ Recovery Systems, des Plaines,Ill., USA). Non-limiting examples of suitable preservation solutionsinclude UW (University of Wisconsin) solution and HTK(histidine-tryptophan-ketoglutarate) solution. During machine perfusion,the perfusion pressure was monitored and verapamil administered asrequired to vasodilate the kidney.

Patients:

After obtaining written, informed consent, 332 patients scheduled forDDRT were randomized 1:1 to receive either a single IV dose of QPI-1002,10.0 mg/kg or isotonic saline placebo (0.9% NaCl) in double-blindfashion, intraoperatively following allograft reperfusion (establishmentof blood flow of the transplanted kidney). 331 patients received studydrug (QPI-1002 or Placebo). Due to a pharmacy dispensing error, onepatient did not receive a study drug.

The following table provides details of patient analysis sets:

TABLE A Analysis Sets ITT (Intent- All patients randomized, transplantedand dosed, to-treat): analyzed as randomized ITTEE: Efficacy evaluable(EE) patients: All patients randomized, transplanted and dosed, analyzedas randomized, (excludes four patients who experi- enced graft loss infirst 24 hrs post-transplant and one patient who did not receive studydrug) - this population was used in the analysis of the primary efficacyendpoint MITT (Modified Same as ITT but analyzed as treated - this popu-intent-to-treat): lation was used in safety analysis mITT(EE): Same asITTEE but analyzed as treated - this popu- lation was used in efficacyanalysis (secondary endpoints)

Key eligibility criteria were designed to enroll patients scheduled toreceive kidney transplant from a deceased donor in 4 subgroups formedbased on the protocol-specified donor type and preservation modality(entirely cold-stored (CS) or at least partially machine-perfused (MP)):

-   -   ECD/CS: ECD kidney that has been preserved by cold storage for        the entire period of cold ischemia time (CIT), regardless of        duration    -   ECD/MP: ECD kidney that has been preserved by machine perfusion        for any interval of time during the period of cold ischemia,        where total CIT has been at least 26 hours    -   SCD/SCD kidney that has been preserved by cold storage where        total CIT has been at least 26 hours    -   SCD kidney that has been preserved by machine perfusion for any        interval of time during the period of cold ischemia, where total        CIT has been at least 26 hours.

Thus, estimated CIT duration did not limit study eligibility in theECD/CS patients study group (Entirely cold stored ECD kidneys).Estimated CIT duration did limit eligibility (>26 hours) in the otherpatient study groups:

The following table provides stratification results for efficacyanalysis:

TABLE B Stratification Results for Efficacy Analysis Strata ITT mITT(EE)ITTEE % of mITT(EE) 332 patients 327 patients 327 patients Overall 100%167 Placebo 163 Placebo 165 Placebo 165 QPI-1002 164 QPI-1002 162QPI-1002 ECD/CS 54.4% 91 Placebo 88 Placebo 89 Placebo 90 QPI-1002 90QPI-1002 88 QPI-1002 ECD/MP 11.6% 18 Placebo 19 Placebo 18 Placebo 20QPI-1002 19 QPI-1002 20 QPI-1002 SCD/CS 11.6% 21 Placebo 21 Placebo 21Placebo 19 QPI-1002 17 QPI-1002 19 QPI-1002 SCD/MP 22.4% 37 Placebo 35Placebo 37 Placebo 36 QPI-1002 38 QPI-1002 35 QPI-1002

It should be noted that organ donor type was not accurately identifieduntil transplant, hence at time of randomization, final organ type wasnot identified. As can be seen from Table B, ECD/CS was largest stratum(N=178 (mITT(EE)), with more than 50% of patients included in thisstratum. ITT & ITTEE strata are presented in Table B as organ donor type(ECD/SCD) used in randomization. mITT(EE) stratum is presented in TableB as per actual organ donor type (ECD/SCD).

The age of the donors was as detailed in following Table C:

TABLE C Donor Age Donor Age (yrs); mean (range) QPI-1002 Placebo OverallPopulation 53.9 (12-84) 53.6 (9-86)  SCD/CS CIT >26 hrs 38.1 (12-56)38.4 (9-59)  SCD/MP CIT >26 hrs 37.1 (12-59) 38.0 (16-59) ECD/CS 62.9(50-84) 62.4 (51-86) ECD/MP CIT >26 hrs 58.5 (50-74) 58.8 (51-69)

DGF was defined as the need for dialysis within 24 hours followingkidney transfer (excluding for hyperkalemia and/or hypervolemia).

Among 327 efficacy-evaluable patients (162 QPI-1002; 165 Placebo), themean ages were 58.9 and 59.1 yrs, respectively; 64.8% and 68.5%, weremale, 23.5% and 22.4% were black (p=ns). There were no significanttreatment differences based on recipient weight, body mass index (BMI),peak pre-transfer % panel reactive antibody (PRA), prior transfusionstatus or HLA mismatching. The pre-transfer DGF risk, determined perIrish nomogram (2010), was 35-36% in both groups (p=ns). Among donors,˜⅔ were ECD and ⅓ SCD in each group; mean CIT and terminal serumcreatinine, % with hypertension and cause of death were notsignificantly different between groups.

Detailed description of recipient and donor demographics is provided inthe following Table D:

TABLE D Stratification Results for Efficacy Analysis Recipients DonorsQPI-1002 Placebo QPI-1002 Placebo Age (yrs); mean (range) 58.9 (24-85) 59.1 (23-83) 53.9 (12-84) 53.6 (9-86)  Sex (% male) 64.8 68.5 58.0 52.1Race (% black) 23.5 22.4 11.7  9.7 Weight*, kg; mean (range) 79.4 79.280.8 (20.79) 82.7 (21.11) BMI**, kg/m²; mean (SD) 27.6 (4.93)  27.6(4.36) — — Prior blood transfusion, n(%)  64 (39.8)  63 (38.2) — — Peak% PRA***, mean (range) 16.9 (0-100) 14.2 (0-99) — — Donor/recipient HLA4.4 (0-6)  4.4 (0-6) — — mismatches, mean (range) *Weight: Recipientweight at Screening evaluation; **BMI: Body mass index ***PRA: Panelreactive antibodies

Table E provides non-demographic donor DGF risk variables:

TABLE E Non-Demographic Donor DGF Risk Variables Donor QPI-1002 PlaceboDonor type: (N = 162) (N = 165) ECD, n (%) 108 (66.7)  108 (65.5)  SCD,n (%) 54 (33.3) 57 (34.5) Cold ischemia time, hrs, mean (range) 22.6(3-65)  23 (5-59) Terminal Scr*, mg/dL; mean (SD) (N = 159) (N = 165)1.2 (0.84) 1.3 (0.95) History of hypertension, n (%) (N = 162) (N = 165)85 (52.5) 90 (54.5) Cause of death (N = 112) (N = 114) Anoxia 30 (26.8)29 (25.4) CVA/stroke 80 (71.4) 84 (73.7) Cardiac 2 (1.8) 1 (0.9) DGFrisk probability, mean (SD) (N = 162) (N = 165) 0.35 (0.16)  0.36(0.18)  In Table E: *Scr: serum creatinine concentration; *PRA: Panelreactive antibodies.

Results

Efficacy Endpoints: The primary endpoint of the study was the incidenceof Delayed Graft Function (DGF) in the Intention-to-Treat (ITT)population of all randomized and transplanted patients, where DGF wasdefined as the need for dialysis initiated within the first 7 dayspost-transplant excluding the following:

(i) Dialysis performed during the first 24 hours for one or more of thefollowing reasons:

-   -   Treatment of hyperkalemia or hypervolemia    -   Hyperacute rejection or other antibody-mediated acute rejection        (biopsy confirmed)    -   Technical vascular complications involving the allograft: renal        arterial and/or venous thrombosis due to vascular injury or        technical surgical complications.

(ii) Dialysis performed during the first 7 days post-transplant for oneor more of the following reasons:

-   -   Obstructive uropathy (Radiographically confirmed)    -   Fulminant recurrence of primary disease (underlying etiology of        ESRD, biopsy confirmed), including focal segmental        glomerulosclerosis    -   A specific diagnosis of thrombotic microangiopathy (Thrombotic        Thrombocytopenic Purpura or Hemolytic-Uremic Syndrome, biopsy        confirmed).

In addition to the primary endpoint, the following key exploratoryefficacy endpoints were also evaluated:

1. The incidence of DGF in the modified Intention-to-Treat ((mITT)(EE))population of all randomized and transplanted patients who receivedstudy drug, where DGF was defined as the need for acute dialysis withinthe first 7 days post-transplant excluding the following:

-   -   Dialysis performed during the first 24 hours for one or more of        the following reasons:        -   Treatment of hyperkalemia or hypervolemia        -   Hyperacute rejection or other antibody-mediated acute            rejection (biopsy confirmed)        -   Technical vascular complications involving the allograft:            renal arterial and/or venous thrombosis due to vascular            injury or technical surgical complications    -   Dialysis performed during the first 7 days post-transplant for        one or more of the following reasons:        -   Obstructive uropathy (Radiographically-confirmed)        -   Fulminant recurrence of primary disease (underlying etiology            of ESRD), including focal segmental glomerulosclerosis        -   A specific diagnosis of thrombotic microangiopathy            (Thrombotic Thrombocytopenic Purpura or Hemolytic-Uremic            Syndrome, biopsy confirmed)

2. Treatment differences in the rate of improvement in renal functionover time

3. Treatment differences in the need for renal replacement therapy.

As defined, DGF occurred in 50 (30.9%) and 60 (36.4%) of QPI-1002 andplacebo patients, respectively (p=0.349), a 15.1% relative reduction inDGF risk. In the largest patient group (ECD/CS, n=178), DGF rates were27.9% and 39.3% for QPI-1002 vs. placebo, respectively, a clinicallymeaningful 30.7% relative reduction (p=0.111). The probability ofremaining dialysis free time-to-first post-transplant dialysis wassignificantly longer improved for QPI-1002 (log-rank p=0.045) and themean duration of dialysis was numerically shorter in this stratum (3.5vs. 9.0 days for QPI-1002 vs. placebo, respectively, p=0.097). Theoverall safety profile of the drug was consistent with that expectedamong DDRT recipients during the early post-transplant period, andsimilar in both treatment groups.

Table F provides primary efficacy endpoint results in ITTEE population(Analyzed as randomized):

TABLE F Primary Efficacy Endpoint (ITTEE population) QPI-1002 PlaceboRelative Risk Strata N DGF n (%) N DGF n (%) Reduction (%) Overall 16250 (30.86) 165 60 (36.36) −15.12 SCD/CS 19  7 (36.84) 21  7 (33.33)10.53 CIT > 26 h SCD/MP 35 11 (31.43) 37 10 (27.03) 16.29 CIT > 26 hECD/CS 88 24 (27.27) 89 35 (39.33) −30.65 ECD/MP 20  8 (40.00) 18  8(44.44) −10 CIT > 26 h

Results for secondary endpoint dialysis received any time during first 7days post-transplant (UNOS definition) in ITTEE population and in ECD/CSstratum are provided in the following Table G:

TABLE G Secondary Endpoint DGF (UNOS Definition) (ITTEE population)ITTEE population overall (N = 327) ECD/CS stratum only (N = 178) DGF DGFN % N % QPI-1002 (n = 164) 62 37.80 QPI-1002 (n = 90) 32 35.56 Placebo(n = 163) 76 46.63 Placebo (n = 88) 43 48.86 Relative −18.9    Relative−27.2    Reduction (%) Reduction (%) Risk difference −8.82 (−19.49,1.84) Risk difference −13.31 (−27.69, 1.08) (95% CI) (95% CI) Fisher'sExact 0.1176 Fisher's Exact 0.0947 p-value p-value

FIGS. 2a and 2b provide results for secondary endpoint probability ofremaining dialysis free time to first post-transplant dialysis inmITT(EE) population (FIG. 2a ) and in ECD/CS stratum (FIG. 2b ), showinga greater reduction improvement in ECD/CS population treated withQP1-1002 as compared to the overall population.

Results for secondary endpoint duration of the initial post-transplantcourse of dialysis in all patients and in ECD/CS stratum are provided inthe following Table H:

TABLE H Secondary Endpoint Duration of the Initial Post-TransplantCourse of Dialysis QPI-1002 Placebo Relative Duration Duration Reduc-(days) (days) tion P- N Mean (SD) N Mean (SD) (%) value* All 51  14.1(27.75) 59  16.9 (30.91) −17 0.620 patients** ECD/ 26 13.40 (34.43) 3421.06 (39.61) −36 0.436 CS*** stratum *P-value: (Unpaired t-test) **Allpatients who received a course of dialysis that began during the firstpost-transplant week

*** All ECD/CS patients who received a course of dialysis that beganduring the first post-transplant week

Table H shows that clinically significant reduction of more than 30% wasachieved in ECD/CS stratum, such that on average one week of dialysiswas saved in patients receiving QPI-1002.

Additional secondary efficacy endpoint was incidence of achieving aneGFR>10 mL/min/1.73 m2 for at least 3 of 7 days during the firstpost-transplant week (non dialysis-based DGF definition). Results areprovided in Table I.

TABLE I Post-transplant Recovery of Renal Function. Incidence ofDecrease in Serum Creatinine ≥10%/day for ≥3 of First 7 Days. Fisher’sQPI-1002 Placebo Relative Exact (n = 107)* (n = 92)* Drug Effect (%)P-value All patients 63 (58.9) 52 (56.5)  4.2 0.775 n (%) (n = 58)* (n =50)* ECD/CS 34 (58.6) 23 (46.0) 27.4 0.247 n (%) *Analysis restricted tothe serum creatinine concentrations of patients who were not receiving acourse of dialysis (and had not received any dialysis for at least 48hours).

Additional non-dialysis DGF secondary efficacy endpoint was slope ofestimated GFR (eGFR) versus time post-transplant. Results are providedin Table J.

TABLE J Non-Dialysis DGF Secondary Efficacy Endpoint. Slope of EstimatedGFR (eGFR) Versus Time Post-transplant. QPI-1002 Placebo eGFR slope*,eGFR slope*, mL/min/1.73 m²/day; mL/min/1.73 m²/day; Relative Effect Nmean (SD) N mean (SD) (%) P-value All patients 4-variable MDRD 164 2.7(3.63) 163 2.4 (3.45) 12.5 0.587 equation Cockroft-Gault 155 2.6 (3.79)153 2.4 (4.18) 8.3 0.701 equation Nankivell equation 100 2.5 (4.01) 1002.6 (4.68) −4.0 0.825 “B” ECD/CS patients 4-variable MDRD 90 2.6 (3.40)88 1.8 (2.88) 44.4 0.065 equation Cockroft-Gault 84 2.6 (3.70) 86 1.7(3.23) 52.9 0.081 equation Nankivell equation 60 2.7 (4.14) 64 2.4(4.42) 12.5 0.633 “B” *eGFR: Calculated after censoring serum creatinineresults obtained during or within 48 hrs of completion of any course ofdialysis.

Additional non-dialysis DGF secondary efficacy endpoint was measured GFRat the day 30 study visit, determined with non-radiolabeled iothalamate,using a commercially available protocol (Mayo Clinic, Rochester Minn.),at North American sites only. Patients who were dialysis dependentwithin 48 hours of the Day 30 visit and those with any history ofallergy to shellfish or iodinated radiocontrast were also excluded fromparticipation in this assay. Results are provided in Table K.

TABLE K Non Dialysis DGF Secondary Efficacy Endpoint. Post-transplantRecovery of Renal Function Measured GFR at the Day 30 Study Visit ITTEEpopulation overall (N = 327) ECD/CS stratum only (N = 178) DGF DGF mGFRmean mGFR mean N (SD) N (SD) QPI-1002 (n = 164) 62 33.8 (31.96) QPI-1002(n = 90) 30 34.8 Placebo (n = 163) 76 29.3 (28.13) Placebo (n = 88) 3621.1 Absolute GFR 4.54 (−5.58, 14.67) Absolute GFR 13.72 (1.02, 26.41)difference (95% difference (95% CI) CI) Fisher's Exact 0.376 Fisher'sExact 0.035 p-value p-value

An additional non-dialysis DGF secondary efficacy endpoint was urineoutput between 2 and 3 days post-transplant, as determined from theprotocol-specified, Day 2-Day 3 quantitated urine output; the totalvolume reported was normalized to that of a 24-hour collection for eachpatient. Analysis excluded results from all patients who received, orwere still receiving dialysis at any time post-transplant through theend of the (Day 2-Day 3) urine collection period. Results are providedin Table L.

TABLE L Non Dialysis DGF Secondary Efficacy Endpoint. Urine OutputBetween 2 and 3 Days Post-transplant Placebo Day 2-Day 3 QPI-1002 urineDay 2-Day 3 output (# urine output (# patients patients >500 ml/Relative >500 ml/day); day) Effect P- N mean (SD) N mean (SD) (%) valueAll 80 71 (88.8) 79 62 (78.5) 13.1 0.090 patients All 40 37 (92.5) 41 28(68.3) 35.4 0.011 ECD/CS patients

Additional non-dialysis DGF secondary efficacy endpoint was based ontreatment differences in the percentages of subjects with DGF, SGF andIGF, as defined per the criteria of Humar et al (ClinicalTransplantation, 2002) and Johnston et al (NDT, 2006). Results areprovided in Table M and the data suggest a shift from DGF to slow graftfunction (SGF) in QPI-1002 treated group.

TABLE M Non Dialysis DGF Secondary Efficacy Endpoint. Incidence ofDelayed, Slow and Immediate Graft Function (EGF, SGF and IGF). QPI-1002Placebo Overall P-value, Adjusted Odds Event Subset (N = 164) (N = 163)(N = 327) unadjusted [2] Ratio [3] Humar et al. All patients (n = 133)(n = 134) (n = 267) IGF 36 (27.1) 36 (26.9) 72 (27.0) 0.112 0.8 (0.5,1.2) SGF 35 (26.3) 22 (16.4) 57 (21.3) DGF 62 (46.6) 76 (56.7) 138(51.7)  Johnston et al All patients (n = 132) (n = 136) (n = 268) IGF 31(23.5) 33 (24.3) 64 (23.9) 0.167 0.8 (0.5, 1.3) SGF 39 (29.5) 27 (19.9)66 (24.6) DGF 62 (47.0) 76 (55.9) 138 (51.5) 

Age Defined Limitations

Primary Efficacy End-Point:

The primary protocol definition-based and UNOS definition-based DGFresults (relative risks and 95% confidence intervals) in the overallmITT(EE) population, the pre-specified ECD/CS stratum and in patientsreceiving kidneys from donors older than 45 or older than 35 years ofage are shown in FIG. 3. FIG. 3 shows a Forest plot demonstrating theimpact of QPI-1002 treatment on DGF relative risk reduction in graftrecipients per donor kidney type. Relative risk (RR) is calculated asthe ratio between DGF incidence in QPI-1002-treated patients andplacebo-treated patients in a given patient subgroup. Box plots:Estimated RR values are marked with “+” inside the boxes. Box sizes areproportional to respective sample sizes. Line ends represent respectivecalculated RR 95% confidence limits (CL). Other definitions: N—number ofpatients in a given subgroup. RRR—relative risk reduction (%) calculatedas RRR (%)=100*(RR-1). LCL—95% lower confidence limit of RRR. UCL—95%upper confidence limit of RRR.

The strongest drug effect was observed in patients receiving kidneysfrom donors older than 45 years of age (n=252; 77% of all evaluablepatients in this study regardless of subgroup attribution) whereQPI-1002 reduced the incidence of DGF by about −30% relative to placebo.The results were the same when either the primary definition of DGF inthis protocol or the widely accepted UNOS definition was used. This −30%difference was both statistically significant (protocol-definedDGF—p=0.048; UNOS DGF definition—p=0.016) and clinically meaningful.Furthermore, when the donor age threshold was lowered to 35 years(n=281; 86% of all evaluable patients in this study regardless ofsubgroup attribution), QPI-1002 still retained its ability to reduce theDGF rate by relative −27%, a clinically and statistically significantdifference (UNOS definition of DGF, p=0.023).

These results are compatible with the new OPTN UNOS classification,which are designed to replace the previous ECD, SCD, DCD classification.

Secondary Efficacy Endpoint:

The impact of QPI-1002 on the severity of DGF was evaluated by lookingat duration (in days) of the initial course of dialysis and intensity(in number of sessions) of dialysis in the first 30 dayspost-transplant. For both parameters, QPI-1002 produced numericallysuperior results in the overall mITT(EE) population (n=110), and in thesubgroups of patients receiving ECD/CS kidneys (n=60) or in patientsreceiving kidneys from older donors at either 45 (n=88) or 35 (n=101)year of age thresholds. Specifically, in dialyzed QPI-1002-treatedpatients receiving ECD/CS kidneys both the duration and the intensity ofdialysis were shorter by −46% relative to the placebo group. In thedialyzed patients receiving older kidneys (whether from donors olderthan 45 or 35 years of age) as well as in the overall patient populationthese reductions ranged from −26% to −33%%.

The impact of QPI-1002 on kidney function in the immediatepost-transplant period (5 days) in non-dialyzed patients was evaluatedby measuring urine output as well as creatinine-based parameters relatedto glomerular function, i.e., eGFR and serum creatinine concentration.The number of patients in whom urine output was greater than 500 ml fromday 2 to day 3 post-transplant was 35% higher among QPI-1002-treatedECD/CS patients when compared to the placebo group (n=81; p=0.011).Among patients receiving kidneys from older donors, the correspondingpercentage increases associated with QPI-1002 treatment were 18% and 20%for donor age thresholds of 35 (n=132) and 45 (n=118) years of age,respectively.

Tables 3a, 3b and 3c show endpoint data for overall recipientpopulation, population of recipients who received a kidney from a donor45 years old and older; and population of recipients who received akidney from a donor 35 years old and older. “Duration of DGF” (.ie, thetotal number of contiguous days counted from the DGF Start date to theDGF Stop date).

TABLE 3a Overall Population Placebo QPI-1002 Absolute Relative Endpoint(N = 163) (N = 164) Difference Difference p-value DGF (Primary Endpoint)N = 163 N = 164 Percent 36.2% 31.1% −5.1 −14.09% 0.351 DGF (UNOS) N =163 N = 164 Percent 46.6% 37.8% −8.8 −18.88% 0.118 Duration of DGF N =59  N = 51  Mean Days (SD) 19.2 (36.9) 14.1 (27.8) −5.13 −26.72% 0.417Intensity of Dialysis N = 59  N = 51  Mean Dialysis Sessions (SD)  8.7(15.7)  6.2 (11.8) −2.48 −28.51% 0.356 24 hr Urine Output >500 ml N =79  N = 80  between Day 2 and Day 3 Percent 78.5% 88.8% 10.3 13.12%0.090 eGFR Slope Cockroft-Gault N = 153 N = 155 Mean Coefficient (SD)2.4 (4.2) 2.6 (3.8) 0.17 7.08% 0.701 eGFR Slope (MDRD) N = 163 N = 164Mean Coefficient (SD) 2.4 (3.5) 2.7 (3.6) 0.21 8.75% 0.587 eGFR SlopeNankivell N = 100 N = 100 Mean Coefficient (SD) 2.6 (4.7) 2.5 (4.0)−0.14 −5.38% 0.825 eGFR Day 30 Cockroft-Gault N = 127 N = 116 Mean (SD)42.3 (20.1) 45.7 (20.2) 3.46 8.18% 0.183 eGFR Day 30 MDRD N = 151 N =146 Mean (SD) 41.4 (20.7) 42.2 (21.2) 0.83 2.00% 0.734 eGFR Day 30Nankivell N = 127 N = 116 Mean (SD) 53.3 (23.2) 56.9 (22.2) 3.54 6.64%0.226 mGFR Day 30 N = 76  N = 62  Mean (SD) 42.6 (21.3) 42.5 (26.5)−0.13 −0.31% 0.974

TABLE 3b >45 years of age Placebo QPI-1002 Absolute Relative Endpoint (N= 163) (N = 164) Difference Difference p-value DGF (Primary Endpoint) N= 127  N = 125 Percent 40.94 28.8 −12.14 −29.66% 0.048 DGF (UNOS) N =127  N = 125 Percent 50.39 35.2 −15.19 −30.15% 0.016 Duration of DGF N =52  N = 36 Mean Days (SD) 20.7 (39.0) 14.3 (32.4) −6.39 −30.87% 0.421Intensity of Dialysis N = 52  N = 36 Mean Dialysis Sessions (SD)  9.3(16.6)  6.2 (13.8) −3.10 −33.3% 0.359 24 hr Urine Output >500 ml N = 60 N = 58 between Day 2 and Day 3 Percent 73.33 87.93 14.6 19.91% 0.063eGFR Slope Cockroft-Gault N = 121  N = 117 Mean Coefficient (SD) 2.0(4.1) 2.4 (3.6) 0.40 20.0% 0.421 eGFR Slope (MDRD) N = 127  N = 125 MeanCoefficient (SD) 2.0 (3.3) 2.6 (3.6) 0.65 32.5% 0.136 eGFR SlopeNankivell N = 83  N = 81 Mean Coefficient (SD) 2.4 (4.6) 2.6 (4.0) 0.197.92% 0.783 eGFR Day 30 Cockroft-Gault N = 101 N = 89 Mean (SD) 39.1(20.4) 42.9 (18.7) 3.81 9.74% 0.183 eGFR Day 30 MDRD N = 117  N = 111Mean (SD) 37.2 (19.6) 40.3 (19.9) 3.12 8.39% 0.235 eGFR Day 30 NankivellN = 101 N = 89 Mean (SD) 50.2 (24.1) 54.9 (21.8) 4.72 8.4% 0.161 mGFRDay 30 N = 56  N = 47 Mean (SD) 39.2 (20.2) 41.1 (23.2) 1.87 4.77% 0.663

TABLE 3c >35 years of age Placebo QPI-1002 Absolute Relative Endpoint (N= 163) (N = 164) Difference Difference p-value DGF (Primary Endpoint) N= 139 N = 142 Percent 41.01 30.99 −10.02 −24.44% 0.084 DGF (UNOS) N =139 N = 142 Percent 51.08 37.32 −13.76 −26.93% 0.023 Duration of DGF N =57  N = 44  Mean Days (SD) 19.6 (37.4) 14.0 (29.6) −5.59 −28.52% 0.419Intensity of Dialysis N = 57  N = 44  Mean Dialysis Sessions (SD)  8.9(15.9)  6.2 (12.7) −2.64 −29.66% 0.370 24 hr Urine Output >500 ml N =64  N = 68  between Day 2 and Day 3 Percent 73.44 86.76 13.32 18.14%0.079 eGFR Slope Cockroft-Gault N = 131 N = 134 Mean Coefficient (SD)2.0 (4.2) 2.5 (3.8) 0.54 27.0% 0.271 eGFR Slope (MDRD) N = 139 N = 142Mean Coefficient (SD) 2.0 (3.3) 2.7 (3.7) 0.66 33.0% 0.115 eGFR SlopeNankivell N = 90  N = 87  Mean Coefficient (SD) 2.3 (4.8) 2.5 (4.0) 0.208.70% 0.767 eGFR Day 30 Cockroft-Gault N = 109 N = 102 Mean (SD) 40.1(20.3) 44.0 (19.2) 3.92 9.78% 0.152 eGFR Day 30 MDRD N = 129 N = 126Mean (SD) 38.5 (19.7) 40.4 (20.0) 1.93 5.01% 0.439 eGFR Day 30 NankivellN = 109 N = 102 Mean (SD) 51.0 (23.7) 55.4 (21.5) 4.45 8.73% 0.156 mGFRDay 30 N = 63  N = 54  Mean (SD) 41.0 (20.9) 42.3 (27.6) 1.33 3.24%0.768

Note that all day 30 GFR values in Tables 3a-3c are observed values andnot change from baseline. Conclusions: The clinical benefit of theQPI-1002 in patients undergoing deceased donor renal transplantationwith marginal kidneys increases with donor age.

The best primary endpoint results were obtained for patients receivingkidneys from donors older than 45 years of age, regardless of theirprimary subgroup classification (that constituted 77% of all theevaluable patients in the study). A threshold of 35 years of age (86% ofall the evaluable patients in the study regardless of subgroupclassification) yielded similar benefits, which are:

Clinically meaningful and, in some subgroups, statistically significantreduction in the incidence of dialysis in the first weekpost-transplant—the primary endpoint of this study.

Clinically meaningful reductions in the duration and intensity ofdialysis in patients who experienced DGF.

Clinically meaningful increases in urine output and eGFR slope over timein the immediate post-transplant period in non-dialyzed patients; and,

Clinically meaningful increase in day 30 eGFR.

Non Dialysis/Renal Function Recovery Efficacy Conclusions:

1. eGFR Slope over time. Overall population showed modest increase inthe rate of eGFR improvement over time in QPI-1002 treated patientscompared to Placebo treated patients. Whereas ECD/CS stratum showed aclinically significant increase (44% MDRD) in the rate of eGFRimprovement over time in QPI-1002 treated patients compared withPlacebo-treated patients (p=0.065 for the eGRF calculated by the MDRDequation).

2. mGRF at Day 30. Overall population showed 4.5 mL/min/1.73 m2 increasein mGFR for QPI-1002 treated patients vs. placebo. Whereas ECD/CSstratum showed a clinically and statistically increase of 13.72mL/min/1.73 m2 in mGFR for QPI-1002 treated patients vs. placebo(p=0.035)

3. Urine output post transplantation. Overall population showed 13%increase in urine output in QPI-1002 patients vs. placebo (p=0.09).Whereas ECD/CS stratum showed 35% increase in urine output in QPI-1002patients vs. placebo. (p=0.011).

4. A shift from DGF to SGF in QPI-1002 treated group.

Conclusion: The rate of DGF was numerically lower among QPI-1002 treatedpatients as compared to patients receiving placebo in the largest(ECD/CS) group and was accompanied by reduced need for post-transferdialysis and a comparable safety profile among both treatment groups.Patients undergoing DDRT with entirely cold-stored ECD kidneys maybenefit from intraoperative, post-reperfusion treatment with QPI-1002 interms of reduced need for dialysis and higher GFRs at 1 monthpost-transfer.

Single-dose treatment with QPI-1002 following vascular reperfusion wasassociated with a safety profile similar to that of Placebo, andcomparable to that expected among recipients of deceased donor renaltransplants.

Overall, recipients of single-dose treatment with QPI-1002 demonstrateda 15% lower rate of DGF when compared to Placebo. This result was notstatistically significant.

Among recipients of entirely cold-stored ECD kidneys, the largeststratum of study patients, a 30% treatment difference in the rate of DGFin favor of QPI-1002 approached statistical significance (p=0.11).

Secondary endpoint analyses demonstrated clinically significantbeneficial treatment differences versus Placebo in the followingendpoints:

-   -   Rate of DGF by the classical UNOS definition    -   Time to first dialysis    -   Severity of DGF: Duration and intensity    -   eGFR Slope    -   Urine output 2-3 days post transplantation    -   Shift from DGF to SGF

For many of these secondary endpoints the results for recipients ofentirely cold-stored ECD kidneys, the largest stratum of study, moreclosely approached (and in some cases achieved) statisticalsignificance.

A potential pharmacoeconomic benefit was observed in the ECD/CS stratumin association with single-dose treatment with QPI-1002, in terms of:

-   -   A significantly higher probability of remaining dialysis free,        as demonstrated by Kaplan-Meier analysis (log-rank p<0.05); and    -   Fewer total dialysis treatments received during the first 30        days post-transplant (lower dialysis “intensity”), a difference        that approached statistical significance (p<0.10).

Example 2: Use of an Inhibitor of a p53 Gene for Prophylaxis of DelayedGraft Function (DGF) in a Recipient of a Kidney from Donors of VariousAges

A study similar to Example 1 was performed to test the effectiveness ofQPI-1002 using kidneys from donors of various ages that did notnecessarily meet the criteria for ECD donors.

In the efficacy analysis, the best primary endpoint results wereobtained for patients receiving kidneys from donors older than 45 yearsof age (regardless of their primary subgroup classification) thatconstituted 77% of all the evaluable patients in the study).Importantly, a threshold of 35 years of age (86% of all the evaluablepatients in the study regardless of subgroup classification) yieldedsimilar benefits (listed below):

Clinically meaningful and, in some subgroups, statistically significantreduction in the incidence of dialysis in the first weekpost-transplant—the primary endpoint of this study.

Clinically meaningful reductions in the duration and intensity ofdialysis in patients who experienced DGF.

Clinically meaningful increases in urine output and eGFR slope over timein the immediate post-transplant period in non-dialyzed patients; and,

Clinically meaningful increase in day 30 eGFR.

Example 3: Dialysis Analysis in Phase II Study

Objective: Dialysis has a pharmacoeconomic impact as well as healthconsequences. Therefore, a comparison of the number of dialyses from day0 to day 180 between treatment groups is of interest.

Statistical Methods: to compare between treatment groups (I5NP, Placebo)

-   -   Zero-inflated Poisson regression model (ZIP), which is a        statistical model based on a Poisson probability distribution        that allows for frequent zero-valued observations. The dialysis        count per patient variable is of such nature.    -   Sensitivity analysis: Non-parametric test: Wilcoxon and Median    -   Descriptive statistics: using counts, mean, standard deviation        (SD), median, minimum and maximum

The analysis was not limited to the first course only or conditioned onDGF event.

Analysis scheme: The analysis was performed on the mitt(EE) population.

-   -   Over all patients    -   Donor age >35 years patient group    -   Donor age >45 years patient group

Study duration analysis

-   -   0-180 day and        -   0-7 days        -   7(+)-30 days        -   30+

Technical (data) details: Dialysis data is drawn out from ‘Dial’ file.In general, in ‘Dial’ data set any record before day 30 is considered asa session and from day 30 and onward the record is regarded as a coursewhich includes the start date and the end date. The number of dialysesin a course is calculated as follow: Number of Dialysis in acourse=3*(Last date of the Course−Start date of a course)/7.

Results:

-   -   Overall patients: Overall, 936 dialysis have been performed in        the study. Their distribution according to time interval and        overall are summarized in Table 4a:

TABLE 4a Overall dialysis distribution and time intervals - totalpopulation Overall Count Count Count dialysis dialysis dialysis Count 0< days <= 0 < days <= 7 < days <= dialysis Treatment 180 7 30 day > 30I5NP 375 (40%) 138 (44%) 113 (41%) 124 (36%) Placebo 561 (60%) 178 (56%)162 (59%) 221 (64%) Total 936 316 275 345 Dialysis

Table 4b summarizes the mean, STD, min, max and median of the number ofdialysis per patients for each treatment group. The most right hand sidecolumns include the p-value obtained from the different tests for thetreatment comparison.

TABLE 4b comparison of mean number of dialysis per patients betweentreatment groups (Total population) Overall: Count dialysis 0 < days <=180 Treatment N mean std min median max MedianP wilcoxonP I5NP 164 2.297.31 0 0 78 0.0857 0.112 Pla- 163 3.44 10.47 0 0 86 cebo

The mean number of dialysis per patients in I5NP group is 2.29, which is−⅔ of the Placebo group (3.44). The p-value is 0.086 according to themedian test.

The p-value obtained by the ZIP model is 0.014, which means that thereis a significant reduction of the number of dialysis per patient in theI5NP group with respect to the Placebo group.

-   -   Donor Age >45 kidneys patients sub-population:    -   Overall, 796 dialysis have been performed in the sub population        of donor age kidneys >45 years. Their distribution according to        time interval and overall are summarized in Table 4c:

TABLE 4c Overall dialysis distribution and time intervals - Donor Age >45 population Donor Age > 45 Count Count Count dialysis dialysisdialysis Count 0 < days <= 0 < days <= 7 < days <= dialysis Treatment180 7 30 day > 30 I5NP 269 (34%)  93 (38%)  65 (30%) 111 (33%) Placebo527 (66%) 151 (62%) 155 (70%) 221 (67%) Total 796 244 220 332 Dialysis

Table 4d contains the descriptive statistics for the mean number ofdialysis per patients according to treatment groups.

TABLE 4d comparison of mean number of dialysis per patients betweentreatment groups Donor Age >45 population Donor Age >45: Count dialysis0 < days <= 180 Treatment n mean std min median max MedianP wilcoxonPI5NP 125 2.15 7.95 0 0 78 0.0076 0.0071 Placebo 127 4.15 11.74 0 1 86

The mean number of dialysis per patients in I5NP group is 2.15, which is˜½ of the Placebo group (4.14). The difference is statisticallysignificant p-value is 0.0076 according to the median test.

The p-value obtained by the ZIP model is <0.01, which means that thereis a significant reduction of the number of dialysis per patient in theI5NP group with respect to the Placebo group.

-   -   Donor Age >35 kidneys patients:        -   Overall, 871 dialysis were performed in the sub population            of donor age kidneys >35 years. Their distribution according            to time interval and overall are summarized in Table 4e:

TABLE 4e Overall dialysis distribution and time intervals - Donor Age >35 population Donor Age > 35 Count Count Count dialysis dialysisdialysis Count 0 < days <= 0 < days <= 7 < days <= dialysis Treatment180 7 30 day > 30 I5NP 322 (37%) 118 (41%)  88 (35%) 116 (34%) Placebo549 (63%) 168 (59%) 160 (65%) 221 (66%) Total 871 286 248 337 Dialysis

Table 4f contains the descriptive statistics for the mean number ofdialysis per patients according to treatment groups.

TABLE 4f comparison of mean number of dialysis per patients betweentreatment groups Donor Age >35 population Donor Age >35: Count dialysis0 < days <= 180 Treatment n mean std min median max MedianP wilcoxonPI5NP 142 2.27 7.63 0 0 78 0.0149 0.0186 Placebo 139 3.95 11.26 0 1 86

The mean number of dialysis per patients in I5NP group is 2.27, which is˜⅗ of the Placebo group result (3.95). The difference is statisticallysignificant p-value is 0.0149 according to the median test.

The p-value obtained by the ZIP model is <0.01, which means that thereis a significant reduction of the number of dialysis per patient in theI5NP group with respect to the Placebo group.

SUMMARY

-   -   In total, the QPI-1002 patient group consumed ˜⅔ of dialysis        than of the Placebo patients group    -   In the overall mITTEE population there is a significant        reduction of the number of dialysis per patient in the I5NP        group with respect to the Placebo group according to the ZIP        regression model, and it is supported by the sensitivity        non-parametric tests.    -   In the sub-population according to donor age the −p-values are        smaller and the difference between treatment groups the mean        numbers of dialysis per patient increase.

Example 4: Generation of Novel Sequences for Active dsNA Compounds

Using proprietary algorithms and the known sequence of the mRNA of thep53 gene (SEQ ID NOS:1-7), the sequences of many potential dsNAcompounds, were generated. The oligonucleotide sequences wereprioritized based on their score in a proprietary algorithm as the bestpredicted sequences for targeting the human p53 gene expression.

Exemplary sense and antisense sequences useful for generating a dsNAtemporary inhibitor of p53 gene are shown in Table 2, supra, and includeSEQ ID NOS:36 and 37; 8 and 21; 9 and 22; 9 and 31; 10 and 23; 11 and24; 12 and 25; 13 and 26; 14 and 27; 15 and 28; 16 and 29; 17 and 30; 18and 31; 18 and 22; 19 and 32; 19 and 28; 20 and 33; and 20 and 21.

Example 5: Identification of Preferred Sequences for Active Nucleic AcidCompounds and Generation of Double-Stranded Nucleic Acid Compounds

The best scoring oligonucleotide sequences were further prioritizedbased on their activity in vitro. For this purpose, dsRNA compounds weresynthesized having the following modification patterns:

dsRNA compounds having unmodified ribonucleotides in the antisensestrand and in the sense strand, and a -dTdT$3′-end overhang in both theantisense strand and the sense strand, with dT designating thymidine anddT$ designating thymidine with no terminal phosphate.

dsRNA compound having alternating 2′-O-methyl (Me) sugar modifiedribonucleotides are present in the first, third, fifth, seventh, ninth,eleventh, thirteenth, fifteenth, seventeenth and nineteenth positions ofthe antisense strand, whereby the very same modification, i.e. a2′-O-Methyl sugar modified ribonucleotides are present in the second,fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth andeighteenth positions of the sense strand.

The following assay was used for the in vitro activity studies.

Activity Assay

About 1.5-2×10⁵ tested human or rat cells endogenously expressing p53genes (Human HCT116 cells or Rat REF52 cells) were grown in 6 wellsplate in 1.5 ml growth medium for about 24 hours to 30-50% confluence.Cells were then transfected with tested dsNA compound in a requiredfinal concentration 0.001-100 nM per well using Liopofectamine 2000reagent. In order to determine the transfection efficiency, of thestudy, 5 wells were treated independently with Lipofectamine 2000reagent and defined as “Negative Control samples” and 5 wells weretransfected independently with active dsRNA at final concentration of 5nM defined as “Control active samples” (positive control). Cy3-labeledsiRNA transfected cells were used as positive control for transfectionefficiency. Cells were then incubated in a 37±1° C., 5% CO₂ incubatorfor 48-72 hours. dsRNA transfected cells were harvested and RNA wasisolated using EZ-RNA kit [Biological Industries (#20-410-100)]. Reversetranscription was performed as follows: cDNA was synthesized and humanand/or rat p53 mRNA levels were determined, accordingly by Real TimeqPCR and normalized to those of the Cyclophilin A (CYNA, PPIA) mRNA foreach sample. dsRNA activity was determined based on the ratio of themRNA quantity in siRNA-treated samples versus non-transfected controlsamples.

As a result of the activity study preferred sequences for novel dsRNAcompounds for down regulation of the p53 gene were identified (resultsnot shown). These sequences are set forth in Table 1, supra (SEQ ID NOS:8-37

Example 6: Generation and Testing of Modified Double-Stranded NucleicAcid Compounds

The preferred sequences (SEQ ID NOS: 8-37) were used for generatingmodified double-stranded nucleic acid compounds. Some modifieddouble-stranded nucleic acid compounds that were generated using thepreferred antisense strand and sense strand sequences are set forth inTables N and O, below. Table P below shows some preferred modifieddouble-stranded nucleic acid compounds that were generated using thepreferred antisense strand and sense strand sequences (SEQ ID NOS:8-37).

TABLE N Duplex Sense Antisense  Name strand (5′ > 3′) strand (5′ > 3′)p53_34 G G A U G U U U G G G A G A U G U A A U U A C A U C U C C C A A AC A U C C p53_35 G A C U C A G A C U G A C A U U C U A U A G A A U G U CA G U C U G A G U C p53_36 G G G U U G G U A G U U U C U A C A A U U G UA G A A A C U A C C A A C C C p53_37 G G G A U G U U U G G G A G A U G UA U A C A U C U C C C A A A C A U C C C p53_38 G G A U C C A C C A A G AC U U G U A U A C A A G U C U U G G U G G A U C C p53_39 G A G G G A U GU U U G G G A G A U A U A U C U C C C A A A C A U C C C U C p53_40 G G GC C U G A C U C A G A C U G A A U U C A G U C U G A G U C A G G C C Cp53_41 G A C U C A G A C U G A C A U U C U U A A G A A U G U C A G U C UG A G U C p53_42 G C A U U U G C A C C U A C C U C A A U U G A G G U A GG U G C A A A U G C p53_43 G G A U G U U U G G G A G A U G U A U A U A CA U C U C C C A A A C A U C C p53_44 G G G C C U G A C U C A G A C U G AU A U C A G U C U G A G U C A G G C C C p53_45 C A G A C C U A U G G A AA C U A C A U G U A G U U U C C A U A G G U C U G

In all tables above and below the duplex names are identified byprefixes “p53” and “TP53” that are used interchangeably.

For all dsRNA compounds in Table N:

A, U, G, C—designates an unmodified ribonucleotide;

A, U, G, C—designates a 2-O-methyl sugar modified ribonucleotide;

In various embodiments, in the nucleic acid compounds in Table N, theribonucleotide at the 3′ terminus and at the 5′ terminus in each of theantisense strand and the sense strand may be phosphorylated ornon-phosphorylated. In some embodiments, of the nucleic acid compoundsin Table N, in each of the antisense strand and the sense strand theribonucleotide at the 3′ terminus is phosphorylated and theribonucleotide at the 5′ terminus is non-phosphorylated. In someembodiments, in each of the nucleic acid compound in Table N, theantisense strand and the sense strand are non-phosphorylated at both the3′ terminus and the 5′ terminus.

Certain exemplary duplexes for generation of double-stranded nucleicacid compounds for down-regulation of a p53 gene are set forth hereinbelow in Table O. Additional duplexes are provided in the Examplessection below.

TABLE O P53 duplexes. Duplex Sense (N′)y Antisense (N)x Name 5→3 5→3p53_13

- C AGACCUAUGGAAAC U A C U-C3-pi AG U AGU

UCC A U A GGUC U G-C3-C3

- C AGACCUAUGGAAA

-C3-pi AG U AGU

UCC A U A GGUC U G-C3-C3

-C A G A C C U A U G G A A A C U A C U-pi A G U A G U U U C C A U A G GU C U G -pi

- C AGACCUAUGGAAA

-C3-pi U G U AGU

UCC A U A GGUC U G-C3-C3

- C AGACCUAUGGAAAC U A C A-C3-pi U G U A G U U U C C A U A G G U C U G-pi

-C A G A C C U A U G G A A A C U A C A-pi UGUAGUUUCCAUAGGUCUG

-C A G A C C U A U G G A A A C U A C A UGUAGUUUCCAUAGGUCUG p53_34

-GGAUGUUUGGGAGA U G U AA-C3-pi U U AC AU

UCC C AAA C A UC C-C3-C3

-GGAUGUUUGGGAGA

-C3-pi U UA C AU

UCC C AAA C A UC C-C3-C3

-GGAUGUUUGGGAGA

-C3-pi AU AC AU

UCC C AAA C A UC C-C3-C3

-GGAUGUUUGGGAGA U G U AU-C3-pi U UA C AU

UCC C AAA C A UC C-C3-C3

-GGAUGUUUGGGAGA

-C3-pi AUA C AU

UCC C AAA C A UC C-C3-C3 GGAUGUUUGGGAGAUGUAUzdTzdT$AUACAUCUCCCAAACAUCCzdTzdT$ p53_35

-GACUCAGACUGACA

-C3-pi U AGAAU

UC AGUCU G AG U C-C3-C3

-GACUCAGACUGA C AU U C U A-C3-pi U AGAA U

U C AGUCU G AG U C-C3-C3

-GACUCAGACUGA C AUU CU A-C3-pi U AGAA

GU C AGUCU G AG U C-C3-C3

-GACUCAGACUGA C A U U CU A-C3-pi U AGAAU

U C AGUCU G AG U C-C3-C3 GACUCAGACUGACAUUCUAzdTzdT$UAGAAUGUCAGUCUGAGUCzdTzdT$ p53_40

-GGGCCUGACUCAGAC U GAA-C3-pi U U C AGU

U GAGU C AGGCCC-C3-C3

-GGGCCUGACUCAGA

-C3-pi U U C AG

C U GAGU C AGGCCC-C3-C3

-GGGCCUGACUCAGAC U GAU-C3-pi U U C AG

CU GAGU C AGGCCC-C3-C3

-GGGCCUGACUCAGA

-C3-pi AU C AG

C U GAGU C AGGCCC-C3-C3

-GGGCCUGACU C AGA CU GAA-C3-pi U U C AG U

U GAGU C AGGCCC-C3-C3

-GGGCCUGACU C AGA CU GAU-C3-pi AU C AG U

U GAGU C AGG CC C-C3-C3

-GGGCCUGACU C AGA CU GAA-C3-pi U U C AG

CU GAGU C AGG CC C-C3-C3 GGGCCUGACUCAGACUGAAzdTzdT$UUCAGUCUGAGUCAGGCCCzdTzdT$ p53_41

-GACUCAGACUGACA

-C3-pi AAGAA U

U C AGUCU G AG U C-C3-C3

-GACUCAGACUGA C AU U C U U-C3-pi AAGAAU

U C AGUCU G AG U C-C3-C3

-GACUCAGACUGA C AUU CU U-C3-pi AAGAAU

UC AGUCU G AG U C-C3-C3

-GACUCAGACUGA C A U U CU U-C3-pi AAGAA

GU C AGUCU G AG U C-C3-C3 GACUCAGACUGACAUUCUUzdTzdT$AAGAAUGUCAGUCUGAGUCzdTzdT$ p53_43

-GGAUGUUUGGGAGA

-C3-pi AU AC AU

UCC C AAA C A UC C-C3-C3

-GGAUGUUUGGGAGA U G U AU-C3-pi A U A C AU

UCC C AAA C A UC C-C3-C3 p53_44

-GGGCCUGACUCAGAC U GAU-C3-pi AU C AGU

U GAGU C AGGCCC-C3-C3

-GGGCCUGACUCAGA

-C3-pi AU C AG

C U GAGU C AGGCCC-C3-C3

-GGGCCUGACU C AGA CU GAU-C3-pi AU C AG

CU GAGU C AGGCCC-C3-C3

For all double-stranded nucleic acid compounds in Table O:

A, U, G, C—designates an unmodified ribonucleotide;

A, U, G, C—designates a 2-O-methyl sugar modified ribonucleotide;

—designates a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond (5′>3′);

cap—designates a capping moiety. In some preferred embodiments thecapping moiety is the group consisting of an abasic ribose moiety, anabasic deoxyribose moiety, an inverted deoxyribose moiety, an inverteddeoxyabasic moiety (idAb), amino-C6 moiety (AM-c6), C6-amino-pi, anon-nucleotide moiety, a mirror nucleotide, a5,6,7,8-tetrahydro-2-naphthalene butyric phosphodiester (THNB) and aconjugate moiety.

pi—designates 3′-phosphate.

z—designates capping moiety

$—designates no terminal phosphate

dT$—designates thymidine (no phosphate)

C3—designates 1,3-Propanediol, mono(dihydrogen phosphate) (C3) [CAS RN:13507-42-1].

C3-C3—designates two consecutive C3 molecules.

In various embodiments of the nucleic acid compounds described in TableO, supra, the C3-C3 non-nucleotide overhang covalently attached at the3′ terminus of the antisense strand is phosphorylated (—C3-C3-pi).

In some embodiments of the nucleic acid compounds described in Table O,supra, in each of the nucleic acid compounds, the ribonucleotide at the5′ terminus in the antisense strand is phosphorylated. In someembodiments of the nucleic acid compounds described in Table O, supra,in each of the nucleic acid compounds, the ribonucleotide at the 5′terminus in the antisense strand is non-phosphorylated.

TABLE P dsRNA Sense (N′)y Antisense (N)x Compound 5-+223 5-+223TP53_13_S2275

- C AGACCUAUGGAAAC U A C U-

-AG U AGU

UCC A U A GGUC U G-

TP53_13_S2276

- C AGACCUAUGGAAAC U A C U-

-AG U AGU

U C C A U A GGUC U G-

TP53_13_S2277

-CAGACCUAUGGAAA

-AGUAGU

UCCAUAGGUCUG-

TP53_13_S2278

- C AGACCUAUGGAAA

-AG U AGU

U C C A U A GGUC U G-

TP53_41_S709 GACUCAGACUGACAUUCUU-

AAGAAUGUCAGUCUGAGUC-

TP53_41_S2279 GACUCAGACUGA C AU U C U U-

-AAGAA U

U C AGUCU G AG U C-

TP53_41_S2298

-GACUCAGACUGACA

-AAGAA U

U C AGUCUGAG U C-

TP53_41_S2299

-GACUCAGACUGACA

-AAGAAU

U C AGUCUGAGUC-

TP53_41_S2300

-GACUCAGACUGA C AU U C U U-

-AAGAAU

U C AGUCUGAGUC-

TP53_44_S2301

-GGGCCUGACUCAGA

-AU C AGU

U GAGU C AGGCCC-

TP53_44_S2302

-GGGCCUGACUCAGA

-AU C AGU

UGAGUCAGGCCC-

TP53_44_S2303

-GGGCCUGACUCAGAC U GAU-

-AU C AGU

U GAGU C AGGCCC-

TP53_44_S2304

-GGGCCUGACUCAGAC U GAU-

-AU C AGU

CUGAGUCAGGCCC-

In all tables above and below the duplex names are identified byprefixes “p53” and “TP53” that are used interchangeably. Thus, forexample a compound identified by prefix “p53_13” and “TP53_13”designates a double-stranded nucleic acid compound having a sense strandsequence 5′ CAGACCUAUGGAAACUACU 3′ (SEQ ID NO:8) and an antisense strandsequence 5′ AGUAGUUUCCAUAGGUCUG 3′ (SEQ ID NO: 21).

For all dsRNA compounds in Table P:

A, U, G, C—designates an unmodified ribonucleotide;

A, U, G, C—designates a 2-O-methyl sugar modified ribonucleotide;

—designates a nucleotide joined to an adjacent nucleotide (5′>3′) by a2′-5′ internucleotide phosphate bond;

C3—designates 1,3-Propanediol, mono(dihydrogen phosphate) alsoidentified as 3-Hydroxypropane-1-phosphate capping moiety [CAS RN:13507-42-1].

C3C3—designates a capping moiety consisting of two consecutive C3molecules

pi—designates 3′-phosphate.

5′-phos—designates 5′-phosphate

These and other chemical modifications may be found in inter alia USpatent and US application publications U.S. Pat. No. 8,362,229;20120283309; 20130035368; 20130324591, to the assignee of the presentapplication and incorporated by reference herein.

Activity of modified double-stranded nucleic acid compounds was studiesin human HCT116 cells and in rat REF52 cells.

Table Q summarizes the in vitro activity results obtained for some ofthe double-stranded nucleic acid molecules in human HCT116 cell line.All of the dsNA compounds are described in Table P, supra.

The p53_13_S500 compound has sense strand SEQ ID NO: 8 and antisensestrand SEQ ID NO: 21 and the following modification pattern: alternating2′-O-methyl (Me) sugar modified ribonucleotides are present in thefirst, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth,seventeenth and nineteenth positions of the antisense strand, wherebythe very same modification, i. e. a 2′-O-Methyl sugar modifiedribonucleotides are present in the second, fourth, sixth, eighth, tenth,twelfth, fourteenth, sixteenth and eighteenth positions of the sensestrand.

The in-vitro activity in Table Q is demonstrated as the % residualtarget mRNA relative to control.

TABLE Q Sample Description Concentration of p53 residual (dsRNA compounddsRNA mRNA % of used) compound control None (control) 100 p53_13_S500 50nM 25 p53_13_S500 25 nM 14 p53_13_S500 5 nM 33 p53_13_S500 1 nM 55p53_1_S500 50 nM 22 p53_1_S500 25 nM 16 p53_1_S500 5 nM 52 p53_1_S500 1nM 107 p53_13_S2275 50 nM 19 p53_13_S2275 25 nM 14 p53_13_S2275 5 nM 25p53_13_S2275 1 nM 60 p53_13_S2276 50 nM 19 p53_13_S2276 25 nM 19p53_13_S2276 5 nM 44 p53_13_S2276 1 nM 112 p53_13_S2277 50 nM 22p53_13_S2277 25 nM 14 p53_13_S2277 5 nM 38 p53_13_S2277 1 nM 112p53_13_S2278 50 nM 41 p53_13_S2278 25 nM 25 p53_13_S2278 5 nM 49p53_13_S2278 1 nM 99 p53_41_S709 50 nM 5 p53_41_S709 25 nM 8 p53_41_S7095 nM 14 p53_41_S709 1 nM 30 p53_41_S2279 50 nM 3 p53_41_S2279 25 nM 8p53_41_S2279 5 nM 3 p53_41_S2279 1 nM 5 p53_41_S2298 50 nM 5p53_41_S2298 25 nM 8 p53_41_S2298 5 nM 5 p53_41_S2299 1 nM p53_41_S229950 nM 3 p53_41_S2299 25 nM 3 p53_41_S2299 5 nM 5 p53_41_S2299 1 nM 5p53_41_S2300 50 nM 3 p53_41_S2300 25 nM 3 p53_41_S2300 5 nM 2p53_41_S2300 1 nM 3 p53_44_S2301 50 nM 5 p53_44_S2301 25 nM 5p53_44_S2301 5 nM 3 p53_44_S2301 1 nM 14 p53_44_S2302 50 nM 3p53_44_S2302 25 nM 3 p53_44_S2302 5 nM 5 p53_44_S2302 1 nM 8p53_44_S2303 50 nM 3 p53_44_S2303 25 nM 5 p53_44_S2303 5 nM 3p53_44_S2303 1 nM 3 p53_44_S2304 50 nM 5 p53_44_S2304 25 nM 5p53_44_S2304 5 nM 11 p53_44_S2304 1 nM 19

Table R summarizes the in vitro activity results obtained for some ofthe double-stranded nucleic acid molecules in rat REF52 cell line.

The in-vitro activity in Table R is demonstrated as the % residualtarget mRNA relative to control.

TABLE R Sample Description Concentration (dsRNA of dsRNA p53 residualmRNA % of compound used) compound control REF52 control None 100p53_13_S500 50 nM 26 25 nM 30 5 nM 63 1 nM 68 p53_41_S709 50 nM 40 25 nM87 5 nM 96 1 nM 211 p53_41_S2279 50 nM 38 25 nM 20 5 nM 21 1 nM 60p53_41_S2298 50 nM 137 25 nM 81 5 nM 71 1 nM 113 p53_41_S2299 50 nM 11625 nM 122 5 nM 107 1 nM 153 p53_41_S2300 50 nM 102 25 nM 81 5 nM 112 1nM 149 p53_44_S2301 50 nM 12 25 nM 14 5 nM 25 1 nM 55 p53_44_S2302 50 nM14 25 nM 9 5 nM 18 1 nM 59 p53_44_S2303 50 nM 20 25 nM 12 5 nM 12 1 nM38 p53_44_S2304 50 nM 22 25 nM 33 5 nM 1 nM

Example 7: Evaluation of the Knock Down Activity of Double-Stranded RNAMolecules Using psiCHECK™-2-System

Three psiCHECK™-2-based (Promega) constructs were prepared for theevaluation of the potential activity. The psiCHECK constructs containedsingle copies of matched complementary guide (AS-CM). 1.3-1.5×10⁶ humanHeLa cells were inoculated in 10 cm dish. Cells were then incubated in37±1° C., 5% CO₂ incubator for 24 hours. Growth medium was replaced oneday post inoculation by 8 ml fresh growth medium and each plate wastransfected with one of the plasmids mentioned above, usingLipofectamine™ 2000 reagent according to manufacturer's protocol andincubated for 5 hours at 37±1° C. and 5% CO₂. Following incubation,cells were re-plated in a 96-well plate at final concentration of 5×10³cells per well in 80 μl growth medium. After 16 hours, cells weretransfected with transfection RNA compound using Lipofectamine™ 2000reagent at final concentrations ranging from 0.01 nM to 100 nM in a 100μl final volume. Cells were then incubated for 48 hours at 37±1° C.following assessment of Renilla and FireFly luciferase activities asdescribed below.

48 hours following transfection with double-stranded RNA compound,Renilla and FireFly luciferase activities were measured in each of thesiRNA transfected samples, using Dual-Luciferase® Assay kit (Promega,Cat#E1960) according to manufacturer procedure. Renilla luciferaseactivity value was divided by Firefly luciferase activity value for eachsample (normalization). Renilla luciferase activity is finally expressedas the percentage of the normalized activity value in tested samplerelative to the normalized value obtained in cells transfected with thecorresponding psiCHECK™-2 plasmid only but with no double-stranded RNA.

The results of the activity study in psiCHECK™-2-System (results notshown) were used to select the best highly active sequences forgenerating highly active double-stranded nucleic acid compound fordown-regulating the p-53 gene.

Although the above examples have illustrated particular ways of carryingout embodiments of the invention, in practice persons skilled in the artwill appreciate alternative ways of carrying out embodiments of theinvention, which are not shown explicitly herein. It should beunderstood that the present disclosure is to be considered as anexemplification of the principles of this invention and is not intendedto limit the invention to the embodiments illustrated.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, equivalents of the specificembodiments of the invention described herein. Such equivalents intendedto be encompassed by the following claims.

1-65. (canceled)
 66. A method of prophylaxis of ischemic reperfusioninjury (IRI) in a kidney at risk of IRI to inhibit acute kidney injury(AKI) in a subject at risk of IRI, the method comprising administeringto the subject a temporary inhibitor of a p53 gene in an amounteffective to provide prophylaxis of AKI in the subject, wherein thesubject is aged 35 years or older; and wherein the inhibitor is asynthetic small interfering ribonucleic acid (siRNA) having thestructure: (antisense strand) (SEQ ID NO: 37) 5′ UGAAGGGUGAAAUAUUCUC 3′(sense strand) (SEQ ID NO: 36) 3′ ACUUCCCACUUUAUAAGAG 5′

wherein each of A, C, U, and G is a ribonucleotide and each consecutiveribonucleotide is joined to the next ribonucleotide by a covalent bond;and wherein alternating ribonucleotides in both the antisense strand andthe sense strand are 2′-O-methyl sugar modified ribonucleotides and a2′-O-methyl sugar modified ribonucleotide is present at both the 5′terminus and the 3′ terminus of the antisense strand and an unmodifiedribonucleotide is present at both the 5′ terminus and the 3′ terminus ofthe sense strand; or a pharmaceutically acceptable salt thereof.
 67. Themethod of claim 66, wherein the subject is aged 45 years or older. 68.The method of claim 66, wherein the subject has had any one or more ofcardiovascular surgery, cardiopulmonary surgery, and renal surgery. 69.The method of claim 67, wherein the subject has had any one or more ofcardiovascular surgery, cardiopulmonary surgery, and renal surgery. 70.The method of claim 66, wherein the subject has had cardiovascularsurgery.
 71. The method of claim 66, wherein the 5′ termini and the 3′termini are unphosphorylated.
 72. The method of claim 66, wherein thepharmaceutically acceptable salt is a sodium salt.
 73. The method claim66, wherein the temporary inhibitor of a p53 gene is administered to thesubject at a dose of about 1 to about 50 mg/kg.
 74. The method of claim66, wherein the temporary inhibitor of a p53 gene is administered to thesubject at a dose of about 10 mg/kg.
 75. The method of claim 66, whereinthe temporary inhibitor is administered to the subject by intravenous(IV) injection.
 76. The method of claim 75, wherein the intravenous (IV)injection is administered to the subject in a single treatment, whereinthe single treatment comprises a single dose or multiple doses.
 77. Themethod of claim 76, wherein the single treatment is a single intravenouspush (IVP).
 78. The method of claim 75, wherein the intravenous (IV)injection is administered to the subject directly into a proximal portof a central venous line or through a peripheral line.
 79. The method ofclaim 66, wherein the temporary inhibitor is conjugated or formulated inliposomes or nanoparticles.
 80. The method of claim 66, wherein thesubject is further administered a medication selected from the groupconsisting of an antiviral agent, an antifungal agent, an antimicrobialagent, and any combination thereof.